Diagnosis and treatment of PTP related disorders

ABSTRACT

The present invention relates to PTP05 polypeptides and PTP10 polypeptides, nucleic acids encoding such polypeptides, cells, tissues and animals containing such nucleic acids, antibodies to such polypeptides, assays utilizing such polypeptides, and methods relating to all of the foregoing. Methods for treatment, diagnosis, and screening are provided for PTP05 and/or PTP10 related diseases or conditions characterized by an abnormal interaction between a PTP05 polypeptide and a PTP05 binding partner and/or a PTP10 polypeptide and a PTP10 binding partner.

RELATED APPLICATIONS

This application claims priority to the U.S. Provisional PatentApplication No. 60/049,756, by Plowman et al., entitled “Diagnosis andTreatment of ALP Related Disorders,” and filed Jun. 11, 1997, which isincorporated herein by reference in its entirety, including anydrawings.

FIELD OF THE INVENTION

The present invention relates to tyrosine phosphatases. In particular,the invention concerns a new family of phosphatase proteins, nucleotidesequences encoding these proteins, various products and assay methodsthat can be used for identifying compounds useful for the diagnosis andtreatment of various PTP-related diseases and conditions, for examplecell proliferative disorders. Two family members have been identifiedwhich we have called PTP05 and PTP10.

BACKGROUND OF THE INVENTION

The following description is provided to aid in understanding theinvention but is not admitted to be prior art to the invention.

Cellular signal transduction is a fundamental mechanism whereby externalstimuli that regulate diverse cellular processes are relayed to theinterior of cells. One of the key biochemical mechanisms of signaltransduction involves the reversible phosphorylation of proteins, whichenables regulation of the activity of mature proteins by altering theirstructure and function. The best characterized protein kinases ineukaryotes phosphorylate proteins on the alcohol moiety of serine,threonine and tyrosine residues. These kinases largely fall into twogroups, those specific for phosphorylating serines and threonines, andthose specific for phosphorylating tyrosines.

The phosphorylation state of a given substrate is also regulated by aclass of proteins responsible for removal of the phosphate group addedto a given substrate by a protein kinase. The protein phosphatases canalso be classified as being specific for either serine/threonine ortyrosine. The known enzymes can be divided into two groups—receptor andnon-receptor type proteins. Most receptor-type protein tyrosinephosphatases (RPTPs) contain two conserved catalytic tyrosinephosphatase domains each of which encompasses a segment of 240 aminoacid residues (Saito et al., Cell Growth and Diff. 2:59-65, 1991). TheRPTPs can be subclassified further based upon the amino acid sequencediversity of their extracellular domains (Saito, et al., supra; Krueger,et al., Proc. Natl. Acad. Sci. USA 89:7417-7421, 1992). Alignment ofprimary peptide sequences of both types of known PTPases shows somesequence consensus in catalytic domains and has made it possible toidentify cDNAs encoding proteins with tyrosine phosphate activity viathe polymerase chain reaction (PCR).

Many kinases and phosphatases are involved in regulatory cascadeswherein their substrates may include, but are not limited to, otherkinases and phosphatases whose activities are regulated by theirphosphorylation state. Ultimately the activity of some downstreameffector is modulated by phosphorylation resulting from activation ofsuch a pathway.

It is well established that the abnormal or inappropriate activity oftyrosine kinases and/or tyrosine phosphatases plays a role in a varietyof human disorders including cell proliferative disorders such ascancer, fibrotic disorders, disorders of the immune system and metabolicdisorders such as diabetes. A need, therefore, exists to identify newtyrosine kinases and phosphatases as a first step in understanding adisease process and the subsequent identification of therapeutictreatments for the disorder.

SUMMARY OF THE INVENTION

Disclosed herein is a family of tyrosine phosphatases expressed inhematopoietic cells, two members of which we have named PTP05 and PTP10.The properties of these phosphatases are described below. The presentinvention concerns PTP05 and PTP10 polypeptides, nucleic acids encodingsuch polypeptides, cells, tissues and animals containing such nucleicacids, antibodies to the polypeptides, assays utilizing thepolypeptides, and methods relating to all of the foregoing.

A first aspect of the invention features an isolated, enriched, orpurified nucleic acid molecule encoding a PTP05 or a PTP10 polypeptide.

By “isolated” in reference to nucleic acid it is meant a polymer of 14,17, 21 or more nucleotides conjugated to each other, including DNA orRNA that is isolated from a natural source or that is synthesized. Theisolated nucleic acid of the present invention is unique in the sensethat it is not found in a pure or separated state in nature. Use of theterm “isolated” indicates that a naturally occurring sequence has beenremoved from its normal cellular (i.e., chromosomal) environment. Thus,the sequence may be in a cell-free solution or placed in a differentcellular environment. The term does not imply that the sequence is theonly nucleotide sequence present, but that it is essentially free (about90-95% pure at least) of non-nucleotide material naturally associatedwith it and thus is meant to be distinguished from isolated chromosomes.

By the use of the term “enriched” in reference to nucleic acid it ismeant that the specific DNA or RNA sequence constitutes a significantlyhigher fraction (2-5) fold of the total DNA or RNA present in the cellsor solution of interest than in normal or diseased cells or in the cellsfrom which the sequence was taken. This could be caused by a person bypreferential reduction in the amount of other DNA or RNA present, or bya preferential increase in the amount of the specific DNA or RNAsequence, or by a combination of the two. However, it should be notedthat “enriched” does not imply that there are no other DNA or RNAsequences present, just that the relative amount of the sequence ofinterest has been significantly increased.

The term “significant” here is used to indicate that the level ofincrease is useful to the person making such an increase, and generallymeans an increase relative to other nucleic acids of about at least 2fold, more preferably at least 5 to 10 fold or even more. The term alsodoes not imply that there is no DNA or RNA from other sources. The othersource DNA may, for example, comprise DNA from a yeast or bacterialgenome, or a cloning vector such as pUC19. This term distinguishes thesequence from naturally occurring enrichment events, such as viralinfection, or tumor type growths, in which the level of one mRNA may benaturally increased relative to other species of mRNA. That is, the termis meant to cover only those situations in which a person has intervenedto elevate the proportion of the desired nucleic acid.

It is also advantageous for some purposes that a nucleotide sequence bein purified form. The term “purified” in reference to nucleic acid doesnot require absolute purity (such as a homogeneous preparation);instead, it represents an indication that the sequence is relativelypurer than in the natural environment (compared to the natural levelthis level should be at least 2-5 fold greater, e.g., in terms ofmg/mL). Individual clones isolated from a cDNA library may be purifiedto electrophoretic homogeneity. The claimed DNA molecules obtained fromthese clones can be obtained directly from total DNA or from total RNA.The cDNA clones are not naturally occurring, but rather are preferablyobtained via manipulation of a partially purified naturally occurringsubstance (messenger RNA). The construction of a cDNA library from mRNAinvolves the creation of a synthetic substance (cDNA) and pureindividual cDNA clones can be isolated from the synthetic library byclonal selection of the cells carrying the cDNA library. Thus, theprocess which includes the construction of a cDNA library from mRNA andisolation of distinct cDNA clones yields an approximately 10 foldpurification of the native message. Thus, purification of at least oneorder of magnitude, preferably two or three orders, and more preferablyfour or five orders of magnitude is expressly contemplated. The term isalso chosen to distinguish clones already in existence which may encodePTP05 or PTP10 but which have not been isolated from other clones in alibrary of clones. Thus, the term covers clones encoding PTP05 or PTP10which are isolated from other non-PTP05 clones or non-PTP10 clones.

The term “nucleic acid molecule” describes a polymer ofdeoxyribonucleotides (DNA) or ribonucleotides (RNA). The nucleic acidmolecule may be isolated from a natural source by cDNA cloning orsubtractive hybridization or synthesized manually. The nucleic acidmolecule may be synthesized manually by the triester synthetic method orby using an automated DNA synthesizer.

The term “cDNA cloning” refers to hybridizing a small nucleic acidmolecule, a probe, to genomic cDNA. The probe hybridizes (binds) tocomplementary sequences of cDNA.

The term “complementary” describes two nucleotides that can formmultiple favorable interactions with one another. For example, adenineis complementary to thymine as they can form two hydrogen bonds.Similarly, guanine and cytosine are complementary since they can formthree hydrogen bonds. Thus if a nucleic acid sequence contains thefollowing sequence of bases, thymine, adenine, guanine and cytosine, a“complement” of this nucleic acid molecule would be a moleculecontaining adenine in the place of thymine, thymine in the place ofadenine, cytosine in the place of guanine, and guanine in the place ofcytosine. Because the complement can contain a nucleic acid sequencethat forms optimal interactions with the parent nucleic acid molecule,such a complement can bind with high affinity to its parent molecule.

The term “hybridize” refers to a method of interacting a nucleic acidsequence with a DNA or RNA molecule in solution or on a solid support,such as cellulose or nitrocellulose. If a nucleic acid sequence binds tothe DNA or RNA molecule with high affinity, it is said to “hybridize” tothe DNA or RNA molecule. The strength of the interaction between theprobing sequence and its target can be assessed by varying thestringency of the hybridization conditions. Under highly stringenthybridization conditions only highly complementary nucleic acidsequences hybridize. Preferably, such conditions prevent hybridizationof nucleic acids having one or two mismatches out of 20 contiguousnucleotides.

Various low or high stringency hybridization conditions may be useddepending upon the specificity and selectivity desired. Stringency iscontrolled by varying salt or denaturant concentrations. Examples ofhybridization conditions are shown in the examples below. High stringentconditions may mean conditions that are at least as stringent as thefollowing: hybridization in 50% formamide, 5×SSC, 50 mM NaH₃PO₄, pH 6.8,0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5×Denhart solutionat 42° C. overnight; washing with 2×SSC, 0.1% SDS at 45° C.; and washingwith 0.2×SSC, 0.1% SDS at 45° C. Those skilled in the art will recognizehow such conditions can be varied to vary specificity and selectivity.

A PTP05 or PTP10 polypeptide can be encoded by a full-length nucleicacid sequence or any portion of the full-length nucleic acid sequence.In preferred embodiments the isolated nucleic acid comprises, consistsessentially of, or consists of a nucleic acid sequence set forth in SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, a nucleic acidsequence that hybridizes to the nucleic acid sequence set forth in SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, or a functionalderivative (as defined below) of either of the foregoing. The nucleicacid may be isolated from a natural source by cDNA cloning orsubtractive hybridization; the natural source may be mammalian (human)blood, semen, or tissue and the nucleic acid may be synthesized by thetriester or other method or by using an automated DNA synthesizer.

In other preferred embodiments, the nucleic acid molecule of theinvention comprises a nucleotide sequence that (a) encodes a polypeptidehaving the full length amino acid sequence set forth in SEQ ID NO:2; (b)is the complement of the nucleotide sequence of (a); (c) hybridizesunder highly stringent conditions to the nucleotide molecule of (a) andencodes a naturally occurring PTP05 or PTP10 polypeptide; (d) encodes aPTP05 or a PTP10 polypeptide having the full length amino acid sequenceof the sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, orSEQ ID NO:8 except that it lacks one or more of the following segmentsof amino acid residues: 1-187, 188-420, 421-426 of SEQ ID NO:5, 44-80,225-457, 458-463 of SEQ ID NO:6, or 1-87, 188-405, 406-412 of SEQ IDNO:7; (e) is the complement of the nucleotide sequence of (d); (f)encodes a polypeptide having the amino acid sequence set forth in SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 from amino acid residues1-187, 188-420, 421-426 of SEQ ID NO:5, 44-80, 225-457, 458-463 of SEQID NO:6, or 1-87, 188-405, 406-412 of SEQ ID NO:7; (g) is the complementof the nucleotide sequence of (f); (h) encodes a polypeptide having thefull length amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, or SEQ ID NO:8 except that it lacks one or more of thedomains selected from the group consisting of an N-terminal domain, acatalytic domain, and a C-terminal domain; or (i) is the complement ofthe nucleotide sequence of (h). The nucleic acid molecule of theinvention is isolated, enriched, or purified from, preferably, a mammal,or most preferably from a human.

In yet other preferred embodiments the nucleic acid is an isolatedconserved or unique region, for example those useful for the design ofhybridization probes to facilitate identification and cloning ofadditional polypeptides, or for the design of PCR probes to facilitatecloning of additional polypeptides.

By “conserved nucleic acid regions”, it is meant regions present on twoor more nucleic acids encoding an PTP05 polypeptide, to which aparticular nucleic acid sequence can hybridize under lower stringencyconditions. Examples of lower stringency conditions suitable forscreening for nucleic acids encoding PTP05 polypeptides are provided inAbe, et al. J. Biol. Chem. 19:13361 (1992) (hereby incorporated byreference herein in its entirety, including any drawings). Preferably,conserved regions differ by no more than 5 out of 20 contiguousnucleotides.

By “unique nucleic acid region” it is meant a sequence present in a fulllength nucleic acid coding for a PTP05 polypeptide or a PTP10polypeptide that is not present in a sequence coding for any other knownnaturally occurring polypeptide. Such regions preferably comprise 14,17, 21 or more contiguous nucleotides present in the full length nucleicacid encoding a PTP05 polypeptide or a PTP10 polypeptide. In particular,a unique nucleic acid region is preferably of human origin.

In yet another aspect, the invention relates to a nucleic acid vectorcomprising a nucleic acid molecule encoding a PTP05 or a PTP10polypeptide and a promoter element effective to initiate transcriptionin a host cell.

The term “nucleic acid vector” relates to a single or double strandedcircular nucleic acid molecule that can be transfected or transformedinto cells and replicate independently or within the host cell genome. Acircular double stranded nucleic acid molecule can be cut and therebylinearized upon treatment with restriction enzymes. An assortment ofvectors, restriction enzymes, and the knowledge of the nucleotidesequences that the restriction enzymes operate upon are readilyavailable to those skilled in the art. A nucleic acid molecule of theinvention can be inserted into a vector by cutting the vector withrestriction enzymes and ligating the two pieces together.

Many techniques are available to those skilled in the art to facilitatetransformation or transfection of the expression construct into aprokaryotic or eukaryotic organism. The terms “transformation” and“transfection” refer to methods of inserting an expression constructinto a cellular organism. These methods involve a variety of techniques,such as treating the cells with high concentrations of salt, an electricfield, or detergent, to render the host cell outer membrane or wallpermeable to nucleic acid molecules of interest.

The term “promoter element” describes a nucleotide sequence that isincorporated into a vector that, once inside an appropriate cell, canfacilitate transcription factor and/or polymerase binding and subsequenttranscription of portions of the vector DNA into mRNA. The promoterelement precedes the 5′ end of the PTP05 or a PTP10 nucleic acidmolecule such that the latter is transcribed into mRNA. Host cellmachinery then translates mRNA into a polypeptide.

Those skilled in the art would recognize that a nucleic acid vector cancontain many other nucleic acid elements besides the promoter elementand the PTP05 or PTP10 nucleic acid molecule. These other nucleic acidelements include, but are not limited to, origins of replication,ribosomal binding sites, nucleic acid sequences encoding drug resistanceenzymes or amino acid metabolic enzymes, and nucleic acid sequencesencoding secretion signals, periplasm or peroxisome localizationsignals, or signals useful for polypeptide purification.

The invention also features a nucleic acid probe for the detection of anucleic acid encoding a PTP05 polypeptide or a PTP10 polypeptide in asample.

The nucleic acid probe contains nucleic acid that will hybridize to asequence of at least 14 contiguous nucleotides set forth in SEQ ID NO:1or SEQ ID NO:2 or a functional derivative thereof. The probe ispreferably at least 14, 17 or more bases in length and selected tohybridize specifically to a unique region of a PTP05 or a PTP10 endocingnucleic acid.

In preferred embodiments the nucleic acid probe hybridizes to nucleicacid encoding at least 14 contiguous amino acids of the sequences setforth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, or afunctional derivative thereof. Various low or high stringencyhybridization conditions may be used depending upon the specificity andselectivity desired. Under highly stringent hybridization conditionsonly highly complementary nucleic acid sequences hybridize. Preferably,such conditions prevent hybridization of nucleic acids having 1 or 2mismatches out of 20 contiguous nucleotides.

Methods for using the probes include detecting the presence or amount ofPTP05 RNA or PTP10 RNA in a sample by contacting the sample with anucleic acid probe under conditions such that hybridization occurs anddetecting the presence or amount of the probe bound to PTP05 RNA orPTP10 RNA. The nucleic acid duplex formed between the probe and anucleic acid sequence coding for a PTP05 polypeptide or a PTP10polypeptide may be used in the identification of the sequence of thenucleic acid detected (for example see, Nelson et al., in NonisotopicDNA Probe Techniques, p. 275 Academic Press, San Diego (Kricka, ed.,1992) hereby incorporated by reference herein in its entirety, includingany drawings). Kits for performing such methods may be constructed toinclude a container having disposed therein a nucleic acid probe.

Another feature of the invention is a nucleic acid molecule as set forthin SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 or fragmentsthereof, comprising one or more regions that encode a PTP05 or a PTP10polypeptide or a PTP05 or a PTP10 domain polypeptide, where the PTP05 orPTP10 polypeptide or the PTP05 or PTP10 domain polypeptide is fused to anon-PTP05 or non-PTP10 polypeptide. Such fused polypeptides include, forexample, but are not limited to, a GST-fusion protein.

The invention also features recombinant nucleic acid, preferably in acell or an organism. The recombinant nucleic acid may contain a sequenceset forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 or afunctional derivative thereof and a vector or a promoter effective toinitiate transcription in a host cell. The recombinant nucleic acid canalternatively contain a transcriptional initiation region functional ina cell, a sequence complimentary to an RNA sequence encoding a PTP05polypeptide or a PTP10 polypeptide and a transcriptional terminationregion functional in a cell.

Another aspect of the invention relates to a recombinant cell or tissuecomprising a nucleic acid molecule encoding a PTP05 or PTO10polypeptide. The recombinant cell may comprise a nucleic acid moleculeencoding either a PTP05 polypeptide; a PTP10 domain polypeptide; or aPTP10 polypeptide or PTP10 domain polypeptide fused to a non-PTP10polypeptide.

The term “recombinant organism” refers to an organism that has a newcombination of genes or nucleic acid molecules. A new combination ofgenes or nucleic acid molecules can be introduced to an organism using awide array of nucleic acid manipulation techniques available to thoseskilled in the art.

The term “organism” relates to any living being comprised of a least onecell. An organism can be as simple as one eukaryotic cell or as complexas a mammal. Therefore, a recombinant organism can also be a recombinantcell, which may be a eukaryotic or a prokaryotic organism.

The term “eukaryote” refers to an organism comprised of cells thatcontain a nucleus. Eukaryotes are differentiated from “prokaryotes”which do not have a nucleus and lack other cellular structures found ineukaryotes, such as mitochondria and endoplasmic reticulum. Prokaryotesinclude unicellular organisms, such as bacteria, while eukaryotes arerepresented by yeast, invertebrates, and vertebrates.

The recombinant cell can harbor a nucleic acid vector that is extragenomic. The term “extragenomic” refers to a nucleic acid vector whichdoes not insert into the cell genome. Many nucleic acid vectors aredesigned with their own origins of replication allowing them to utilizethe recombinant cell replication machinery to copy and propagate thevector nucleic acid sequence. These vectors are small enough that theyare not likely to harbor nucleic acid sequences homologous to genomicsequences of the recombinant cell. Thus these vectors replicateindependently of the host genome and do not recombine with or integrateinto the genome.

A recombinant cell can harbor a portion of a nucleic acid vector in anintra genomic fashion. The term “intragenomic” defines a nucleic acidconstruct that is incorporated within the cell genome. Multiple nucleicacid vectors available to those skilled in the art contain nucleic acidsequences that are homologous to nucleic acid sequences in a particularorganism's genomic DNA. These homologous sequences will result inrecombination events that integrate portions of the vector into thegenomic DNA. Those skilled in the art can control which nucleic acidsequences of the vector are integrated into the cell genome by flankingthe portion to be incorporated into the genome with homologous sequencesin the vector.

Another aspect of the invention features an isolated, enriched, orpurified PTP05 polypeptide or an isolated, enriched, or purified PTP10polypeptide.

By “PTP05 polypeptide” or “PTP10 polypeptide” it is meant an amino acidsequence substantially similar to the sequence shown in SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or fragments thereof. A sequencethat is substantially similar will preferably have at least 90% identity(more preferably at least 95% and most preferably 99-100%) to thesequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

The PTP05 or PTP10 polypeptides of the present invention preferably havea substantially similar biological activity to the protein encoded bythe full length nucleic acid sequence set forth in SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, or SEQ ID NO:4 or to SEQ ID NO:1 the proteins withamino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,or SEQ ID NO:8. By “biological activity” it is meant an activity of thePTP10 protein in a cell. The biological activity of the PTP10 is relatedto some of the activities of the cell which include, but are not limitedto, cell proliferation motogenesis, metastasis, tumor escape, celladhesion, transformation, or apoptosis.

By “identity” is meant a property of sequences that measures theirsimilarity or relationship. Identity is measured by dividing the numberof identical residues in the two sequences by the total number ofresidues and multiplying the product by 100. Thus, two copies of exactlythe same sequence have 100% identity, but sequences that are less highlyconserved and have deletions, additions, or replacements have a lowerdegree of identity. Those skilled in the art will recognize that severalcomputer programs are available for determining sequence identity.

By “isolated” in reference to a polypeptide is meant a polymer of 6, 12,18 or more amino acids conjugated to each other, including polypeptidesthat are isolated from a natural source or that are synthesized. Theisolated polypeptides of the present invention are unique in the sensethat they are not found in a pure or separated state in nature. Use ofthe term “isolated” indicates that a naturally occurring sequence hasbeen removed from its normal cellular environment. Thus, the sequencemay be in a cell-free solution or placed in a different cellularenvironment. The term does not imply that the sequence is the only aminoacid chain present, but that it is essentially free (about 90-95% pureat least) of material naturally associated with it.

By the use of the term “enriched” in reference to a polypeptide it ismeant that the specific amino acid sequence constitutes a significantlyhigher fraction (2-5 fold) of the total of amino acids present in thecells or solution of interest than in normal or diseased cells or in thecells from which the sequence was taken. This could be caused by aperson by preferential reduction in the amount of other amino acidsequences present, or by a preferential increase in the amount of thespecific amino acid sequence of interest, or by a combination of thetwo. However, it should be noted that “enriched” does not imply thatthere are no other amino acid sequences present, just that the relativeamount of the sequence of interest has been significantly increased.

The term “significant” here is used to indicate that the level ofincrease is useful to the person making such an increase, and generallymeans an increase relative to other amino acid sequences of about atleast 2 fold, more preferably at least 5 to 10 fold or even more. Theterm also does not imply that there are no amino acid sequences fromother sources. The other source amino acid may, for example, compriseamino acid encoded by a yeast or bacterial genome, or a cloning vectorsuch as pUC19. The term is meant to cover only those situations in whicha person has intervened to elevate the proportion of the desired nucleicacid.

It is also advantageous for some purposes that an amino acid sequence bein purified form. The term “purified” in reference to a polypeptide doesnot require absolute purity (such as a homogeneous preparation);instead, it represents an indication that the sequence is relativelypurer than in the natural environment (compared to the natural levelthis level should be at least 2-5 fold greater, e.g., in terms ofmg/mL). Purification of at least one order of magnitude, preferably twoor three orders, and more preferably four or five orders of magnitude isexpressly contemplated. The substance is preferably free ofcontamination at a functionally significant level, for example 90%, 95%,or 99% pure.

In another aspect the invention features an isolated, enriched, orpurified PTP05 polypeptide fragment or a PTP10 polypeptide fragment.

By “a PTP05 polypeptide fragment” or “PTP10 polypeptide fragment” it ismeant an amino acid sequence that is less than the full-length aminoacid sequence. The full-length amino acid sequences of three PTP05isoforms are shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7. Examplesof fragments include PTP05 domains, PTP05 mutants and PTP05-specificepitopes and PTP10 domains, PTP10 mutants and PTP10-specific epitopes.

By “a PTP05 domain” or a “PTP10 domain” it is meant a portion of thePTP05 polypeptide or the PTP10 polypeptide having homology to amino acidsequences from one or more known proteins wherein the sequence predictssome common function, interaction or activity. Well known examples ofdomains are the SH2 (Src Homology 2) domain (Sadowski, et al., Mol.Cell. Biol. 6:4396, 1986; Pawson and Schlessinger, Curr. Biol. 3:434,1993), the SH3 domain (Mayer, et al., Nature 332:272, 1988; Pawson andSchlessinger, Curr. Biol. 3:434, 1993), and pleckstrin (PH) domain(Ponting, TIBS 21:245, 1996; Haslam, et al., Nature 363:309, 1993), allof which are domains that mediate protein:protein interaction orprotein:lipid interaction, and the kinase catalytic domain (Hanks andHunter, FASEB J. 9:576-595, 1995). Computer programs designed to detectsuch homologies are well known in the art. The relative homology is atleast 20%, more preferably at least 30% and most preferably at least35%.

By a “PTP05 mutant” or “PTP10 mutant” it is meant a PTP05 polypeptide orPTP10 polypeptide which differs from the native sequence in that one ormore amino acids have been changed, added or deleted. Changes in aminoacids may be conservative or non-conservative. By “conservative” it ismeant the substitution of an amino acid for one with similar propertiessuch as charge, hydrophobicity, structure, etc. Examples of polypeptidesencompassed by this term include, but are not limited to, (1) chimericproteins which comprise a portion of a PTP05 polypeptide or PTP10polypeptide sequence fused to a non-PTP05 or non-PTP10 polypeptidesequence, for example a polypeptide sequence of hemagglutinin (HA), (2)PTP05 or PTP10 proteins lacking a specific domain, for example thecatalytic domain, and (3) PTP05 or PTP10 proteins having a pointmutation. A PTP05 mutant or PTP10 mutant will retain some usefulfunction such as, for example, binding to a natural binding partner,catalytic activity, or the ability to bind to a PTP05 specific antibodyor a PTP10 specific antibody (as defined below).

By “PTP05-specific epitope” or “PTP10-specific epitope” it is meant asequence of amino acids that is both antigenic and unique to PTP05 or toPTP10, respectively. A PTP05-specific epitope can be used to producePTP05-specific antibodies and a PTP10-specific epitope can be used toproduce PTP10-specific antibodies, as more fully described below.Particularly preferred epitopes a shown in the Examples section below.

By “recombinant PTP05 polypeptide” or “recombinant PTP10 polypeptide” itis meant to include a polypeptide produced by recombinant DNA techniquessuch that it is distinct from a naturally occurring polypeptide eitherin its location (e.g., present in a different cell or tissue than foundin nature), purity or structure. Generally, such a recombinantpolypeptide will be present in a cell in an amount different from thatnormally observed in nature.

The polypeptide of the invention comprises an amino acid sequence having(a) the full length amino acid sequence set forth in SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, or SEQ ID NO:8; (b) the full length amino acidsequence of the sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, or SEQ ID NO:8, except that it lacks one or more of the followingsegments of amino acid residues: 1-187, 188-420, 421-426 of SEQ ID NO:5,44-80, 225-457, 458-463 of SEQ ID NO:6, or 1-87, 188-405, 406-412 of SEQID NO:7; (c) the amino acid sequence set forth in SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, or SEQ ID NO:8 from amino acid residues 1-187,188-420, 421-426 of SEQ ID NO:5, 44-80, 225-457, 458-463 of SEQ ID NO:6,or 1-87, 188-405, 406-412 of SEQ ID NO:7; or (d) the full length aminoacid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQID NO:8 except that it lacks one or more of the domains selected fromthe group consisting of an N-terminal domain, a catalytic domain, and aC-terminal domain.

In yet another aspect the invention features an antibody (e.g., amonoclonal or polyclonal antibody) having specific binding affinity to aPTP05 or PTP10 polypeptide or PTP05 or PTP10 polypeptide fragment. By“specific binding affinity” is meant that the antibody binds to target(PTP05 or PTO10) polypeptides with greater affinity than it binds toother polypeptides under specified conditions. Antibodies havingspecific binding affinity to a PTP05 or a PTP10 polypeptide may be usedin methods for detecting the presence and/or amount of a PTP05 or PTP10polypeptide in a sample by contacting the sample with the antibody underconditions such that an immunocomplex forms and detecting the presenceand/or amount of the antibody conjugated to the PTP05 or PTP10polypeptide. Diagnostic kits for performing such methods may beconstructed to include a first container containing the antibody and asecond container having a conjugate of a binding partner of the antibodyand a label, such as, for example, a radioisotope. The diagnostic kitmay also include notification of an FDA approved use and instructionstherefor.

The term “polyclonal” refers to antibodies that are heterogenouspopulations of antibody molecules derived from the sera of animalsimmunized with an antigen or an antigenic functional derivative thereof.For the production of polyclonal antibodies, various host animals may beimmunized by injection with the antigen. Various adjuvants may be usedto increase the immunological response, depending on the host species.

“Monoclonal antibodies” are substantially homogenous populations ofantibodies to a particular antigen. They may be obtained by anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. Monoclonal antibodies may be obtainedby methods known to those skilled in the art. See, for example, Kohler,et al., Nature 256:495-497 (1975), and U.S. Pat. No. 4,376,110.

The term “antibody fragment” refers to a portion of an antibody, oftenthe hypervariable region and portions of the surrounding heavy and lightchains, that displays specific binding affinity for a particularmolecule. A hypervariable region is a portion of an antibody thatphysically binds to the polypeptide target.

In another aspect the invention features a hybridoma which produces anantibody having specific binding affinity to a PTP05 polypeptide or ahybridoma which produces an antibody having specific binding affinity toa PTP10 polypeptide. By “hybridoma” is meant an immortalized cell linewhich is capable of secreting an antibody, for example a PTP05 antibodyor a PTP10 antibody. In preferred embodiments the PTP05 antibodycomprises a sequence of amino acids that is able to specifically bind aPTP05 polypeptide, and the PTP10 antibody comprises a sequence of aminoacids that is able to specifically bind a PTP10 polypeptide.

The invention features a method for identifying human cells containing aPTP05 polypeptide, a PTP10 polypeptide or a related sequence. The methodinvolves identifying the novel polypeptide in human cells usingtechniques that are routine and standard in the art, such as thosedescribed herein for identifying PTP05 (e.g., cloning, Southern orNorthern blot analysis, in situ hybridization, PCR amplification, etc.).

The invention also features methods of screening cells for naturalbinding partners of PTP05 polypeptides or PTP10 polypeptides. By“natural binding partner” it is meant a protein that interacts withPTP05 or with PTP10. Binding partners include ligands, agonists,antagonists and downstream signaling molecules such as adaptor proteinsand may be identified by techniques well known in the art such asco-immunoprecipitation or by using, for example, a two-hybrid screen.(Fields and Song, U.S. Pat. No. 5,283,173, issued Feb. 1, 1994 and,incorporated be reference herein.) The present invention also featuresthe purified, isolated or enriched versions of the polypeptidesidentified by the methods described above.

In another aspect, the invention provides a method for identifying asubstance capable of modulating PTO05 or PTP10 activity comprising thesteps of (a) contacting a PTO05 or PTP10 polypeptide with a testsubstance; and (b) determining whether the substance alters the activityof said polypeptide.

The invention also features another method of identifying substancescapable of modulating the function of a PTO05 or PTP10 polypeptide. Themethod comprises the following steps: (a) expressing a PTO05 or PTP10polypeptide in cells; (b) adding a compound to the cells; and (c)monitoring a change or an absence of a change in cell phenotype, cellproliferation, catalytic activity of the PTO05 or PTP10 polypeptide, andbinding a natural binding partner.

The term “compound” includes small organic molecules including, but notlimited to, oxindolinones, quinazolines, tyrphostins, quinoxalines, andthose contained within extracts from natural sources. Examples of suchcompounds are included in section XII, below.

The term “function” refers to the cellular role of a serine-threonineprotein kinase. The serine-threonine protein kinase family includesmembers that regulate many steps in signaling cascades, includingcascades controlling cell growth, migration, differentiation, geneexpression, muscle contraction, glucose metabolism, cellular proteinsynthesis, and regulation of the cell cycle.

The term “modulates” refers to the ability of a compound to alter thefunction of a protein kinase. A modulator preferably activates thecatalytic activity of a protein kinase, more preferably activates orinhibits the catalytic activity of a protein kinase depending on theconcentration of the compound exposed to the protein kinase, or mostpreferably inhibits the catalytic activity of a protein kinase.

The term “catalytic activity,” in the context of the invention, definesthe ability of a protein kinase to phosphorylate a substrate. Catalyticactivity can be measured, for example, by determining the amount of asubstrate converted to a product as a function of time. Phosphorylationof a substrate occurs at the active-site of a protein kinase. Theactive-site is normally a cavity in which the substrate.

The term “substrate” as used herein refers to a molecule that isphoshorylated by or directly interacts with the protein kinase. Thesubstrate is preferably a peptide and more preferably a protein. Forexample, in relation to the protein kinase RAF, preferred substrates areMEK and the MEK substrate MAPK.

The term “activates” refers to increasing the cellular function of aprotein kinase. The protein kinase function is preferably theinteraction with a natural binding partner or catalytic activity.

The term “inhibit” refers to decreasing the cellular function of aprotein kinase. The protein kinase function is preferably theinteraction with a natural binding partner or catalytic activity.

The term “modulates” also refers to altering the function of a proteinkinase by increasing or decreasing the probability that a complex formsbetween a protein kinase and a natural binding partner. A modulatorpreferably increases the probability that such a complex forms betweenthe protein kinase and the natural binding partner, more preferablyincreases or decreases the probability that a complex forms between theprotein kinase and the natural binding partner depending on theconcentration of the compound exposed to the protein kinase, and mostpreferably decreases the probability that a complex forms between theprotein kinase and the natural binding partner.

The term “complex” refers to an assembly of at least two molecules boundto one another. Signal transduction complexes often contain at least twoprotein molecules bound to one another, either transiently or insuccession. For instance, a receptor protein tyrosine kinase, GRB2, SOS,and RAF sequentially interact in response to a mitogenic ligand.

The term “expressing” as used herein refers to the production of a PTO05or PTP10 polypeptide from a nucleic acid vector containing a PTO05 orPTP10 gene within a cell. The nucleic acid vector is transfected intocells using well known techniques in the art as described herein.

The term “adding” as used herein refers to administering a solutioncomprising a compound to the medium bathing cells. The solutioncomprising the compound can also comprise an agent, such as dimethylsulfoxide, which facilitates the uptake of the compound into the cells.

The term “monitoring” refers to observing the effect of adding thecompound to the cells of the method. The effect can be manifested in achange in cell phenotype, cell proliferation, protein kinase catalyticactivity, or in the interaction between a protein kinase and a naturalbinding partner.

The term “cell phenotype” refers to the outward appearance of a cell ortissue or the function of the cell or tissue. Examples of cell or tissuephenotype are cell size (reduction or enlargement), cell proliferation(increased or decreased numbers of cells), cell differentiation (achange or absence of a change in cell shape), cell survival, apoptosis(cell death), or the utilization of a metabolic nutrient (e.g., glucoseuptake). Change or the absence of change in cell phenotype is readilymeasured by techniques known in the art.

The term “cell proliferation” refers to the rate at which a group ofcells divides. The number of cells growing in a vessel can bequantitated by a person skilled in the art when that person visuallycounts the number of cells in a defined area using a common lightmicroscope. Alternatively, cell proliferation rates can be quantitatedby laboratory apparatae that optically measure the density of cells inan appropriate medium.

The method can utilize any of the molecules disclosed in the invention.These molecules include nucleic acid molecules encoding PTO05 or PTP10polypeptides, nucleic acid vectors, recombinant cells, polypeptides, orantibodies of the invention.

In a preferred embodiment, the invention provides a method for treatingor preventing an abnormal condition by administering a compound which isa modular of PTO05 or PTP10 function in vitro. The abnormal conditionpreferably involves abnormality in PTO05 or PTP10 signal transductionpathway, and most preferably is cancer. Such compounds preferably showpositive results in one or more in vitro assays for an activitycorresponding to treatment of the disease or disorder in question (suchas the assays described in Example 6 below). Examples of substances thatcan be screened for favorable activity are provided in section XIIbelow.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF FIGURES

FIG. 1 shows a computer generated alignment between the nucleic acid(FIG. 1A) and amino acid (FIG. 1B) sequences from the catalytic domainsof PTP05 and PTP10. As can be seen from the alignments, PTP05 and PTP10are highly related at both the nucleic acid (92%) and amino acid (85%)level, suggesting they are members of a family of PTPs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a new family of tyrosine phosphatasesand the isolation and characterization of two members of this family,proteins which we have called PTP05 and PTP10, nucleotide sequencesencoding PTP05 and PTP10, various products and assay methods that can beused to identify compounds useful for the diagnosis and treatment ofvarious PTP05 and PTP10 related diseases and conditions, for examplediabetes. Polypeptides derived from PTP05 and PTP10 and nucleic acidsencoding such polypeptides may be produced using well known and standardsynthesis techniques when given the sequences presented herein.

PTP05 is a tyrosine phosphatase with an apparent molecular weight ofapproximately 49 kDa. Two additional isoforms have been identified, onelarger (approximately 54 kDa) and one smaller (approximately 47 kDa).Primary sequence analysis shows that PTP05 is comprised of threedomains: an N-terminal domain, a catalytic domain, and a C-terminaldomain. The lack of a hydrophobic stretch of amino acids generallycharacterized as a transmembrane region indicates that PTP05 is anon-receptor tyrosine phosphatase. PTP10 is also a tyrosine phosphatasewith significant homology to PTP05. Together they define a new family ofPTPs.

The polypeptide and nucleotide sequences of the invention can be used,for example, to generate antibodies for use as diagnostic kits, or tocreate recombinant cell lines that can be used to identify modulators ofPTP05 or PTP10 activity. Moreover, the sequences of the invention can beused to obtain full-length sequences of PTP05 and/or PTP10 fromadditional species, in particular humans, using techniques well known inthe art and also described below.

I. Nucleic Acids Encoding PTP05 and/or PTP10 Polypeptides

A first aspect of the invention features nucleic acid sequences encodinga PTP05 polypeptide or a PTP10 polypeptide. Included within the scope ofthis invention are the functional equivalents of the herein-describedisolated nucleic acid molecules. Functional equivalents or derivativescan be obtained in several ways. The degeneracy of the genetic codepermits substitution of certain codons by other codons which specify thesame amino acid and hence would give rise to the same protein. Thenucleic acid sequence can vary substantially since, with the exceptionof methionine and tryptophan, the known amino acids can be coded for bymore than one codon. Thus, portions or all of the PTP05 gene could besynthesized to give a nucleic acid sequence significantly different fromthat shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. Likewiseportions or all of the PTP10 gene could be synthesized to give a nucleicacid sequence significantly different from that shown in SEQ ID NO:4.The encoded amino acid sequence thereof would, however, be preserved.

In addition, the nucleic acid sequence may comprise a nucleotidesequence which results from the addition, deletion or substitution of atleast one nucleotide to the 5′-end and/or the 3′-end of the nucleic acidformula shown in SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, or SEQ ID NO:4,or a derivative thereof. Any nucleotide or polynucleotide may be used inthis regard, provided that its addition, deletion or substitution doesnot alter the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ IDNO:7, or SEQ ID NO:8 which is encoded by the nucleotide sequence. Forexample, the present invention is intended to include any nucleic acidsequence resulting from the addition of ATG as an initiation codon atthe 5′-end of a PTP05 or PTP10 nucleic acid sequence or its functionalderivative, or from the addition of TTA, TAG or TGA as a terminationcodon at the 3′-end of the inventive nucleotide sequence or itsderivative. Moreover, the nucleic acid molecule of the present inventionmay, as necessary, have restriction endonuclease recognition sites addedto its 5′-end and/or 3′-end.

Such functional alterations of a given nucleic acid sequence afford anopportunity to promote secretion and/or processing of heterologousproteins encoded by foreign nucleic acid sequences fused thereto. Allvariations of the nucleotide sequence of the PTP05 genes, PTP10 genesand fragments thereof permitted by the genetic code are, therefore,included in this invention.

Further, it is possible to delete codons or to substitute one or morecodons by codons other than degenerate codons to produce a structurallymodified polypeptide, but one which has substantially the same utilityor activity of the polypeptide produced by the unmodified nucleic acidmolecule. As recognized in the art, the two polypeptides arefunctionally equivalent, as are the two nucleic acid molecules whichgive rise to their production, even though the differences between thenucleic acid molecules are not related to degeneracy of the geneticcode.

Functional equivalents or derivatives of PTP05 or PTP10 can also beobtained using nucleic acid molecules encoding one or more functionaldomains of the PTP05 polypeptide or the PTP10 polypeptide, respectively.For example, the catalytic domain of PTP05 functions to remove phosphatemolecules bound onto tyrosine residues and a nucleic acid sequenceencoding the catalytic domain alone or linked to other heterologousnucleic acid sequences can be considered a functional derivative ofPTP05. Other functional domains of these proteins include, but are notlimited to, the proline-rich region within the N-terminal domain, andthe C-terminal domain. Nucleic acid sequences encoding these domains areshown in SEQ ID NO:1 as follows: N-terminal domain approximately199-759; catalytic domain approximately 760-1458, C-terminal domainapproximately 1459-1476.

II. A Nucleic Acid Probe for the Detection of PTP05 or PTP10

A nucleic acid probe of the present invention may be used to probe anappropriate chromosomal or cDNA library by usual hybridization methodsto obtain another nucleic acid molecule of the present invention. Achromosomal DNA or cDNA library may be prepared from appropriate cellsaccording to recognized methods in the art (e.g. “Molecular Cloning: ALaboratory Manual”, second edition, edited by Sambrook, Fritsch, &Maniatis, Cold Spring Harbor Laboratory, 1989).

In the alternative, chemical synthesis is carried out in order to obtainnucleic acid probes having nucleotide sequences which correspond topUN-terminal and C-terminal portions of the amino acid sequence of thepolypeptide of interest. Thus, the synthesized nucleic acid probes maybe used as primers in a polymerase chain reaction (PCR) carried out inaccordance with recognized PCR techniques, essentially according to PCRProtocols, “PCR Protocols, A Guide to Methods and Applications”, editedby Innis et al., Academic Press, 1990, utilizing the appropriatechromosomal or cDNA library to obtain the fragment of the presentinvention.

One skilled in the art can readily design such probes based on thesequence disclosed herein using methods of computer alignment andsequence analysis known in the art (e.g. “Molecular Cloning: ALaboratory Manual”, second edition, edited by Sambrook, Fritsch, &Maniatis, Cold Spring Harbor Laboratory, 1989). The hybridization probesof the present invention can be labeled by standard labeling techniquessuch as with a radiolabel, enzyme label, fluorescent label,biotin-avidin label, chemiluminescence, and the like. Afterhybridization, the probes may be visualized using known methods.

The nucleic acid probes of the present invention include RNA as well asDNA probes and nucleic acids modified in the sugar, phosphate or eventhe base portion as long as the probe still retains the ability tospecifically hybridize under conditions as disclosed herein. Such probesare generated using techniques known in the art. The nucleic acid probemay be immobilized on a solid support. Examples of such solid supportsinclude, but are not limited to, plastics such as polycarbonate, complexcarbohydrates such as agarose and sepharose, acrylic resins, such aspolyacrylamide and latex beads, and nitrocellulose. Techniques forcoupling nucleic acid probes to such solid supports are well known inthe art.

The test samples suitable for nucleic acid probing methods of thepresent invention include, for example, cells or nucleic acid extractsof cells, or biological fluids. The sample used in the above-describedmethods will vary based on the assay format, the detection method andthe nature of the tissues, cells or extracts to be assayed. Methods forpreparing nucleic acid extracts of cells are well known in the art andcan be readily adapted in order to obtain a sample which is compatiblewith the method utilized.

III. A Probe Based Method and Kit For Detecting PTP05 or PTP10

One method of detecting the presence of PTP05 or PTP10 in a samplecomprises (a) contacting the sample with one of the above-describednucleic acid probes, under conditions such that hybridization occurs,and (b) detecting the presence of the probe bound to a nucleic acidmolecule in the sample. One skilled in the art would select the nucleicacid probe according to techniques known in the art as described above.Samples to be tested include but should not be limited to RNA samples ofhuman tissue.

A kit for detecting the presence of PTP05 or PTP10 in a sample comprisesat least one container having disposed therein an above-describednucleic acid probe. The kit may further comprise other containerscomprising one or more of the following: wash reagents and reagentscapable of detecting the presence of bound nucleic acid probe. Examplesof detection reagents include, but are not limited to radiolabelledprobes, enzymatically labeled probes (horseradish peroxidase, alkalinephosphatase), and affinity labeled probes (biotin, avidin, orsteptavidin).

In detail, a compartmentalized kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers or strips of plastic or paper. Suchcontainers allow the efficient transfer of reagents from one compartmentto another compartment such that the samples and reagents are notcross-contaminated and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the probe or primers used in the assay,containers which contain wash reagents (such as phosphate bufferedsaline, Tris-buffers, and the like), and containers which contain thereagents used to detect the hybridized probe, bound antibody, amplifiedproduct, or the like. One skilled in the art will readily recognize thatthe nucleic acid probes described in the present invention can readilybe incorporated into one of the established kit formats which are wellknown in the art.

IV. DNA Constructs Comprising a PTP05 Nucleic Acid Molecule or PTP10Nucleic Acid Molecule and Cells Containing these Constructs

The present invention also relates to a recombinant DNA moleculecomprising, 5′ to 3′, a promoter effective to initiate transcription ina host cell and one of the above-described nucleic acid molecules. Inaddition, the present invention relates to a recombinant DNA moleculecomprising a vector and a nucleic acid molecule described herein. Thepresent invention also relates to a nucleic acid molecule comprising atranscriptional region functional in a cell, a sequence complimentary toan RNA sequence encoding an amino acid sequence corresponding to a PTP05polypeptide, a PTP10 polypeptide or functional derivative of either, anda transcriptional termination region functional in said cell. Theabove-described molecules may be isolated and/or purified DNA molecules.

The present invention also relates to a cell or organism that contains aPTP05 nucleic acid molecule or a PTP10 nucleic acid molecule, asdescribed herein, and thereby is capable of expressing a peptide. Thepolypeptide may be purified from cells which have been altered toexpress the polypeptide. A cell is said to be “altered to express adesired polypeptide” when the cell, through genetic manipulation, ismade to produce a protein which it normally does not produce or whichthe cell normally produces at lower levels. One skilled in the art canreadily adapt procedures for introducing and expressing either genomic,cDNA, or synthetic sequences into either eukaryotic or prokaryoticcells.

A nucleic acid molecule, such as DNA, is said to be “capable ofexpressing” a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are “operably linked” to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene sequence expression. Theprecise nature of the regulatory regions needed for gene sequenceexpression may vary from organism to organism, but will in generalinclude a promoter region which, in prokaryotes, contains both thepromoter (which directs the initiation of RNA transcription) as well asthe DNA sequences which, when transcribed into RNA, will signalsynthesis initiation. Such regions will normally include those5′-non-coding sequences involved with initiation of transcription andtranslation, such as the TATA box, capping sequence, CAAT sequence, andthe like.

If desired, the non-coding region 3′ to the sequence encoding a PTP05gene or a PTP10 gene may be obtained by the above-described cloningmethods. This region may be retained for its transcriptional terminationregulatory sequences, such as termination and polyadenylation. Thus, byretaining the 3′-region naturally contiguous to the DNA sequenceencoding a PTP05 gene or a PTP10 gene, the transcriptional terminationsignals may be provided. Where the transcriptional termination signalsare not satisfactorily functional in the expression host cell, then a 3′region functional in the host cell may be substituted.

Two DNA sequences (such as a promoter region sequence and a PTP05sequence) are said to be operably linked if the nature of the linkagebetween the two DNA sequences does not (1) result in the introduction ofa frame-shift mutation, (2) interfere with the ability of the promoterregion sequence to direct the transcription of the second sequence, forexample a PTP05 gene sequence, or (3) interfere with the ability of thesecond sequence to be transcribed by the promoter region sequence. Thus,a promoter region would be operably linked to a DNA sequence if thepromoter were capable of effecting transcription of that DNA sequence.Thus, transcriptional and translational signals recognized by anappropriate host are necessary to express a PTP05 gene or PTP10 gene.

The present invention encompasses the expression of a PTP05 gene, aPTP10 gene (or a functional derivative thereof) in either prokaryotic oreukaryotic cells. Prokaryotic hosts are, generally, very efficient andconvenient for the production of recombinant proteins and are,therefore, one type of preferred expression system for these genes.Prokaryotes most frequently are represented by various strains of E.coli. However, other microbial strains may also be used, including otherbacterial strains.

In prokaryotic systems, plasmid vectors that contain replication sitesand control sequences derived from a species compatible with the hostmay be used. Examples of suitable plasmid vectors may include pBR322,pUC118, pUC119 and the like; suitable phage or bacteriophage vectors mayinclude lgt10, lgt11 and the like; and suitable virus vectors mayinclude pMAM-neo, pKRC and the like. Preferably, the selected vector ofthe present invention has the capacity to replicate in the selected hostcell.

Recognized prokaryotic hosts include bacteria such as E. coli and thosefrom genera such as Bacillus, Streptomyces, Pseudomonas, Salmonella,Serratia, and the like. However, under such conditions, the polypeptidewill not be glycosylated. The prokaryotic host must be compatible withthe replicon and control sequences in the expression plasmid.

To express PTP05 or PTP10 (or a functional derivative thereof) in aprokaryotic cell, it is necessary to operably link the gene sequence toa functional prokaryotic promoter. Such promoters may be eitherconstitutive or, more preferably, regulatable (i.e., inducible orderepressible). Examples of constitutive promoters include the intpromoter of bacteriophage 1, the bla promoter of the b-lactamase genesequence of pBR322, and the CAT promoter of the chloramphenicol acetyltransferase gene sequence of pPR325, and the like. Examples of inducibleprokaryotic promoters include the major right and left promoters ofbacteriophage l (P_(L) and P_(R)), the trp, recA, lacZ, lacI, and galpromoters of E. coli, the a-amylase (Ulmanen et al., J. Bacteriol.162:176-182, 1985) and the sigma-28-specific promoters of B. subtilis(Gilman et al., Gene Sequence 32:11-20(1984)), the promoters of thebacteriophages of Bacillus (Gryczan, In: The Molecular Biology of theBacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters(Ward et al., Mol. Gen. Genet. 203:468-478, 1986). Prokaryotic promotersare reviewed by Glick (J. Ind. Microbiot. 1:277-282, 1987); Cenatiempo(Biochimie 68:505-516, 1986); and Gottesman (Ann. Rev. Genet.18:415-442, 1984).

Proper expression in a prokaryotic cell also requires the presence of aribosome binding site upstream of the gene sequence-encoding sequence.Such ribosome binding sites are disclosed, for example, by Gold et at.(Ann. Rev. Microbiol. 35:365-404, 1981). The selection of controlsequences, expression vectors, transformation methods, and the like, aredependent on the type of host cell used to express the gene.

As used herein, “cell”, “cell line”, and “cell culture” may be usedinterchangeably and all such designations include progeny. Thus, thewords “transformants” or “transformed cells” include the primary subjectcell and cultures derived therefrom, without regard to the number oftransfers. It is also understood that all progeny may not be preciselyidentical in DNA content, due to deliberate or inadvertent mutations.However, as defined, mutant progeny have the same functionality as thatof the originally transformed cell.

Host cells which may be used in the expression systems of the presentinvention are not strictly limited, provided that they are suitable foruse in the expression of the PTP peptide of interest. Suitable hosts mayoften include eukaryotic cells. Preferred eukaryotic hosts include, forexample, yeast, fungi, insect cells, and mammalian cells, either in vivoor in tissue culture. Mammalian cells which may be useful as hostsinclude HeLa cells, cells of fibroblast origin such as VERO, 3T3 orCHO-K1, or cells of lymphoid origin (such as 32D cells) and theirderivatives. Preferred mammalian host cells include SP2/0 and J558L, aswell as neuroblastoma cell lines such as IMR 332 and PC12 which mayprovide better capacities for correct post-translational processing.

In addition, plant cells are also available as hosts, and controlsequences compatible with plant cells are available, such as thecauliflower mosaic virus 35S and 19S, and nopaline synthase promoter andpolyadenylation signal sequences. Another preferred host is an insectcell, for example the Drosophila larvae. Using insect cells as hosts,the Drosophila alcohol dehydrogenase promoter can be used. Rubin,Science 240:1453-1459, 1988). Alternatively, baculovirus vectors can beengineered to express large amounts of PTP05 or PTP10 in insects cells(Jasny, Science 238:1653, 1987); Miller et al., In: Genetic Engineering(1986), Setlow, J. K., et al., eds., Plenum, Vol. 8, pp. 277-297).

Any of a series of yeast gene sequence expression systems can beutilized which incorporate promoter and termination elements from theactively expressed gene sequences coding for glycolytic enzymes areproduced in large quantities when yeast are grown in mediums rich inglucose. Known glycolytic gene sequences can also provide very efficienttranscriptional control signals. Yeast provides substantial advantagesin that it can also carry out post-translational peptide modifications.A number of recombinant DNA strategies exist which utilize strongpromoter sequences and high copy number of plasmids which can beutilized for production of the desired proteins in yeast. Yeastrecognizes leader sequences on cloned mammalian gene sequence productsand secretes peptides bearing leader sequences (i.e., pre-peptides). Fora mammalian host, several possible vector systems are available for theexpression of PTP05 or PTP10.

A particularly preferred yeast expression system is that utilizingSchizosaccharmocyces pombe. This system is useful for studying theactivity of members of the Src family (Superti-Furga, et al, EMBO J.12:2625, 1993) and other non-receptor-TKs, the function of which isoften regulated by the activity of tyrosine phosphatases.

A wide variety of transcriptional and translational regulatory sequencesmay be employed, depending upon the nature of the host. Thetranscriptional and translational regulatory signals may be derived fromviral sources, such as adenovirus, bovine papilloma virus,cytomegalovirus, simian virus, or the like, where the regulatory signalsare associated with a particular gene sequence which has a high level ofexpression. Alternatively, promoters from mammalian expression products,such as actin, collagen, myosin, and the like, may be employed.Transcriptional initiation regulatory signals may be selected whichallow for repression or activation, so that expression of the genesequences can be modulated. Of interest are regulatory signals which aretemperature-sensitive so that by varying the temperature, expression canbe repressed or initiated, or are subject to chemical (such asmetabolite) regulation.

Expression of PTP05 or PTP10 in eukaryotic hosts requires the use ofeukaryotic regulatory regions. Such regions will, in general, include apromoter region sufficient to direct the initiation of RNA synthesis.Preferred eukaryotic promoters include, for example, the promoter of themouse metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen.1:273-288, 1982); the TK promoter of Herpes virus (McKnight, Cell31:355-365, 1982); the SV40 early promoter (Benoist et al., Nature(London) 290:304-310, 1981); the yeast gal4 gene sequence promoter(Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975, 1982);Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955, 1984).

Translation of eukaryotic mRNA is initiated at the codon which encodesthe first methionine. For this reason, it is preferable to ensure thatthe linkage between a eukaryotic promoter and a DNA sequence whichencodes PTP05 or PTP10 (or a functional derivative thereof) does notcontain any intervening codons which are capable of encoding amethionine (i.e., AUG). The presence of such codons results either inthe formation of a fusion protein (if the AUG codon is in the samereading frame as the coding sequence) or a frame-shift mutation (if theAUG codon is not in the same reading frame as a PTP05 coding sequence).

A PTP05 nucleic acid molecule or PTP10 nucleic acid molecule and anoperably linked promoter may be introduced into a recipient prokaryoticor eukaryotic cell either as a nonreplicating DNA (or RNA) molecule,which may either be a linear molecule or, more preferably, a closedcovalent circular molecule (a plasmid). Since such molecules areincapable of autonomous replication, the expression of the gene mayoccur through the transient expression of the introduced sequence.Alternatively, permanent or stable expression may occur through theintegration of the introduced DNA sequence into the host chromosome.

A vector may be employed which is capable of integrating the desiredgene sequences into the host cell chromosome. Cells which have stablyintegrated the introduced DNA into their chromosomes can be selected byalso introducing one or more markers which allow for selection of hostcells which contain the expression vector. The marker may provide forprototropy to an auxotrophic host, biocide resistance, e.g.,antibiotics, or heavy metals, such as copper, or the like. Theselectable marker gene sequence can either be directly linked to the DNAgene sequences to be expressed, or introduced into the same cell byco-transfection. Additional elements may also be needed for optimalsynthesis of single chain binding protein mRNA. These elements mayinclude splice signals, as well as transcription promoters, enhancers,and termination signals. cDNA expression vectors incorporating suchelements include those described by Okayama, Mol. Cell. Bio. 3:280,1983.

The introduced nucleic acid molecule can be incorporated into a plasmidor viral vector capable of autonomous replication in the recipient host.Any of a wide variety of vectors may be employed for this purpose.Factors of importance in selecting a particular plasmid or viral vectorinclude: the ease with which recipient cells that contain the vector maybe recognized and selected from those recipient cells which do notcontain the vector; the number of copies of the vector which are desiredin a particular host; and whether it is desirable to be able to“shuttle” the vector between host cells of different species.

Preferred prokaryotic vectors include plasmids such as those capable ofreplication in E. coli (such as, for example, pBR322, ColEl, pSC101,pACYC 184, pVX. Such plasmids are, for example, disclosed by Sambrook(cf. “Molecular Cloning: A Laboratory Manual”, second edition, edited bySambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, (1989)).Bacillus plasmids include pC194, pC221, pT127, and the like. Suchplasmids are disclosed by Gryczan (In: The Molecular Biology of theBacilli, Academic Press, NY (1982), pp. 307-329). Suitable Streptomycesplasmids include p1J101 (Kendall et al., J. Bacteriol. 169:4177-4183,1987), and streptomyces bacteriophages such as fC31 (Chater et al., In:Sixth International Symposium on Actinomycetales Biology, AkademiaiKaido, Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids arereviewed by John et al. (Rev. Infect. Dis. 8:693-704, 1986), and Izaki(Jpn. J. Bacteriol. 33:729-742, 1978).

Preferred eukaryotic plasmids include, for example, BPV, vaccinia, SV40,2-micron circle, and the like, or their derivatives. Such plasmids arewell known in the art (Botstein et al., Miami Wntr. Symp. 19:265-274,1982); Broach, In: “The Molecular Biology of the Yeast Saccharomyces:Life Cycle and Inheritance”, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., p. 445-470 (1981); Broach, Cell 28:203-204, 1982); Bollonet at., J. Clin. Hematol. Oncol. 10:39-48, 1980); Maniatis, In: CellBiology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression,Academic Press, NY, pp. 563-608 (1980).

Once the vector or nucleic acid molecule containing the construct(s) hasbeen prepared for expression, the DNA construct(s) may be introducedinto an appropriate host cell by any of a variety of suitable means,i.e., transformation, transfection, conjugation, protoplast fusion,electroporation, particle gun technology, calciumphosphate-precipitation, direct micro injection, and the like. After theintroduction of the vector, recipient cells are grown in a selectivemedium, which selects for the growth of vector-containing cells.Expression of the cloned gene molecule(s) results in the production ofPTP05, PTP10 or fragments or functional derivatives thereof of either.This can take place in the transformed cells as such, or following theinduction of these cells to differentiate (for example, byadministration of bromodeoxyuracil to neuroblastoma cells or the like).A variety of incubation conditions for the transformed cells can be usedto foster expression of the polypeptides of the present invention. Themost preferred conditions are those which mimic physiologicalconditions.

V. PTP05 and PTP10 Polypeptides

Also a feature of the invention are PTP05 and PTP10 polypeptides. Avariety of methodologies known in the art can be utilized to obtain thepolypeptides of the present invention. They may be purified from tissuesor cells which naturally produce them. Alternatively, theabove-described isolated nucleic acid sequences can be used to expressthe proteins of the invention recombinantly.

Any eukaryotic organism can be used as a source for the polypeptide ofthe invention, as long as the source organism naturally contains such apolypeptide. As used herein, “source organism” refers to the originalorganism from which the amino acid sequence is derived, regardless ofthe organism the protein is expressed in and ultimately isolated from.

One skilled in the art can readily follow known methods for isolatingproteins in order to obtain the peptide free of natural contaminants.These include, but are not limited to: size-exclusion chromatography,HPLC, ion-exchange chromatography, and immuno-affinity chromatography.

A PTP05 and PTP10 proteins, like all proteins, are comprised of distinctfunctional units or domains. In eukaryotes, proteins sorted through theso-called vesicular pathway (bulk flow) usually have a signal sequence(also called a leader peptide) in the N-terminus, which is cleaved offafter the translocation through the ER (endoplasmic reticulum) membrane.Some N-terminal signal sequences are not cleaved off, remaining astransmembrane segments, but it does not mean these proteins are retainedin the ER; they can be further sorted and included in vesicles.Non-receptor proteins generally function to transmit signals within thecell, either by providing sites for protein:protein interactions or byhaving some catalytic activity (contained within a catalytic domain),often both. Methods of predicting the existence of these various domainsare well known in the art. Protein:protein interaction domains can beidentified by comparison to other proteins. The SH2 domain, for exampleis a protein domain of about 100 amino acids first identified as aconserved sequence region between the proteins Src and Fps (Sadowski, etal., Mol. Cell. Bio. 6:4396, 1986). Similar sequences were later foundin many other intracellular signal-transducing proteins. SH2 domainsfunction as regulatory modules of intracellular signaling cascades byinteracting with high affinity to phosphotyrosine-containing proteins ina sequence specific and strictly phosphorylation-dependent manner (Mayerand Baltimore, Trends Cell. Biol. 3:8, 1993). Kinase or phosphatasecatalytic domains can be identified by comparison to other knowncatalytic domains with kinase or phosphatase activity. See, for exampleHanks and Hunter, FASEB J. 9:576-595, 1995.

Primary sequence analysis of the PTP05 amino acid sequence (shown in SEQID NO:5 with isoforms shown in SEQ ID NO:6 and SEQ ID NO:7) reveals thatit and its isoforms do not contain a signal sequence or transmembranedomain, and it is, therefore, an intracellular protein. Comparison toknown protein sequences revels that PTP05 is comprised of several uniquedomains. These include a 187 amino acid N-terminal domain (shown fromamino acid number 1-187 of SEQ ID NO:5), a 242 amino acid catalyticdomain (shown from amino acid number 188-420 of SEQ ID NO:5), and a 5amino acid C-terminal domain (shown from amino acid number 421-426 ofSEQ ID NO:5).

Two additional isoforms of PTP05 were also identified, a “long” form(SEQ ID NO:6) and a “C-trunc” form (SEQ ID NO:7). The “long” form has a37 amino acid insertion in the N-terminal domain (amino acids 44-80 ofSEQ ID NO:6) which extends this domain to 224 amino acids. The catalyticdomain extends from amino acid 225-457 of SEQ ID NO:6 and the C-terminaldomain extents from amino acids 458-463 of SEQ ID NO:6. The “C-trunc”form results from a deletion of nucleotides 1415-1507 of SEQ ID NO:1,most likely due to alternative exon splicing. This deletion results in areplacement of the C-terminal 21 amino acids with a unique 7 amino acidsequence. This change eliminates a conserved C-terminal portion of thecatalytic domain, which may affect enzymatic activity. The N-terminaldomain of the “C-trunc” form extends from amino acid 1-87 of SEQ IDNO:7, the catalytic domain from amino acids 188-405 of SEQ ID NO:7 andthe unique C-terminal domain from 406-412 of SEQ ID NO:7.

The domains of these proteins have a variety of uses. An example of sucha use is to make a polypeptide consisting of a PTP05 catalytic domainand a heterologous protein such as glutathione S-transferase (GST). Sucha polypeptide can be used in a biochemical assay for PTP05 catalyticactivity useful for studying PTP05 substrate specificity or foridentifying substances that can modulate PTP05 catalytic activity.Alternatively, one skilled in the art could create a PTP05 polypeptidelacking at least one of the three major domains. Such a polypeptide,when expressed in a cell, is able to form complexes with the naturalbinding partner(s) of PTP05 but unable to transmit any signal furtherdownstream into the cell, ie. it would be signaling incompetent and thuswould be useful for studying the biological relevance of PTP05 activity.(See, as an example, Gishizky, et al., PNAS: 10889, 1995).

VI. An Antibody Having Binding Affinity to a PTP05 Polypeptide, anAntibody Having Binding Affinity to a PTP10 Polypeptide and HybridomasProducing these Antibodies

The present invention also relates to antibodies having specific bindingaffinity to a PTP05 polypeptide or to a PTP10 polypeptide. Thepolypeptide may have the amino acid sequence set forth in SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or be a fragment thereof, or atleast 6 contiguous amino acids thereof. Such an antibody may beidentified by comparing its binding affinity to the desired polypeptide,for example a PTP05 polypeptide, with its binding affinity to another(non-PTP05) polypeptide. Those which bind selectively to the desiredpolypeptide would be chosen for use in methods requiring a distinctionbetween the desired polypeptide and other polypeptides. Such methodscould include, but should not be limited to, the analysis of alteredexpression of the desired polypeptide in tissue containing otherpolypeptides and assay systems using whole cells.

A PTP05 polypeptide or PTP10 polypeptide of the present invention can beused to produce antibodies or hybridomas. One skilled in the art willrecognize that if an antibody is desired, such a peptide would begenerated as described herein and used as an immunogen. Preferred PTP05or PTP10 peptides in this respect are shown in the Examples sectionbelow. The antibodies of the present invention include monoclonal andpolyclonal antibodies, as well fragments of these antibodies, andhumanized forms. Humanized forms of the antibodies of the presentinvention may be generated using one of the procedures known in the artsuch as chimerization or CDR grafting. The present invention alsorelates to a hybridoma which produces the above-described monoclonalantibody, or binding fragment thereof. A hybridoma is an immortalizedcell line which is capable of secreting a specific monoclonal antibody.

In general, techniques for preparing monoclonal antibodies andhybridomas are well known in the art (Campbell, “Monoclonal AntibodyTechnology: Laboratory Techniques in Biochemistry and MolecularBiology,” Elsevier Science Publishers, Amsterdam, The Netherlands, 1984;St. Groth et al., J. Immunol. Methods 35:1-21, 1980). Any animal (mouse,rabbit, and the like) which is known to produce antibodies can beimmunized with the selected polypeptide. Methods for immunization arewell known in the art. Such methods include subcutaneous orintraperitoneal injection of the polypeptide. One skilled in the artwill recognize that the amount of polypeptide used for immunization willvary based on the animal which is immunized, the antigenicity of thepolypeptide and the site of injection.

The polypeptide may be modified or administered in an adjuvant in orderto increase the peptide antigenicity. Methods of increasing theantigenicity of a polypeptide are well known in the art. Such proceduresinclude coupling the antigen with a heterologous protein (such asglobulin or b-galactosidase) or through the inclusion of an adjuvantduring immunization.

For monoclonal antibodies, spleen cells from the immunized animals areremoved, fused with myeloma cells, such as SP2/0-Agl4 myeloma cells, andallowed to become monoclonal antibody producing hybridoma cells. Any oneof a number of methods well known in the art can be used to identify thehybridoma cell which produces an antibody with the desiredcharacteristics. These include screening the hybridomas with an ELISAassay, western blot analysis, or radioimmunoassay (Lutz, et al., Exp.Cell Res. 175:109-124, 1988). Hybridomas secreting the desiredantibodies are cloned and the class and subclass is determined usingprocedures known in the art (Campbell, “Monoclonal Antibody Technology:Laboratory Techniques in Biochemistry and Molecular Biology”, supra,1984).

For polyclonal antibodies, antibody containing antisera is isolated fromthe immunized animal and is screened for the presence of antibodies withthe desired specificity using one of the above-described procedures. Theabove-described antibodies may be detectably labeled. Antibodies can bedetectably labeled through the use of radioisotopes, affinity labels(such as biotin, avidin, and the like), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, and the like) fluorescentlabels (such as FITC or rhodamine, and the like), paramagnetic atoms,and the like. Procedures for accomplishing such labeling are well-knownin the art, for example, see (Stemberger, et al., J. Histochem.Cytochem. 18:315, 1970; Bayer, et al., Meth. Enzym. 62:308, 1979;Engval, et al., Immunot. 109:129, 1972; Goding, J. Immunol. Meth.13:215, 1976). The labeled antibodies of the present invention can beused for in vitro, in vivo, and in in situ assays to identify cells ortissues which express a specific peptide.

The above-described antibodies may also be immobilized on a solidsupport. Examples of such solid supports include plastics such aspolycarbonate, complex carbohydrates such as agarose and sepharose,acrylic resins and such as polyacrylamide and latex beads. Techniquesfor coupling antibodies to such solid supports are well known in the art(Weir et al., “Handbook of Experimental Immunology” 4th Ed., BlackwellScientific Publications, Oxford, England, Chapter 10, 1986; Jacoby etal., Meth. Enzym. 34, Academic Press, N.Y., 1974). The immobilizedantibodies of the present invention can be used for in vitro, in vivo,and in situ assays as well as in immunochromotography.

Furthermore, one skilled in the art can readily adapt currentlyavailable procedures, as well as the techniques, methods and kitsdisclosed above with regard to antibodies, to generate peptides capableof binding to a specific peptide sequence in order to generaterationally designed antipeptide peptides, for example see Hurby et al.,“Application of Synthetic Peptides: Antisense Peptides”, In SyntheticPeptides, A User's Guide, W.H. Freeman, NY, pp. 289-307(1992), andKaspczak et al., Biochemistry 28:9230-8(1989).

VII. An Antibody Based Method and Kit for Detecting PTP05 or PTP10

The present invention encompasses a method of detecting a PTP05polypeptide or a PTP10 polypeptide in a sample comprising incubating atest sample with one or more of the antibodies of the present inventionand determining whether the antibody binds to the test sample. Themethod can include the steps of, for example: (a) contacting the samplewith an above-described antibody, under conditions such thatimmunocomplexes form, and (b) detecting the presence of said antibodybound to the polypeptide. Altered levels, either an increase ordecrease, of PTP05 or PTP10 in a sample as compared to normal levels mayindicate an abnormality or disorder.

Conditions for incubating an antibody with a test sample vary.Incubation conditions depend on the format employed in the assay, thedetection methods employed, and the type and nature of the antibody usedin the assay. One skilled in the art will recognize that any one of thecommonly available immunological assay formats (such asradioimmunoassays, enzyme-linked immunosorbent assays, diffusion basedOuchterlony, or rocket immunofluorescent assays) can readily be adaptedto employ the antibodies of the present invention. Examples of suchassays can be found in Chard, “An Introduction to Radioimmunoassay andRelated Techniques” Elsevier Science Publishers, Amsterdam, TheNetherlands (1986); Bullock et al., “Techniques in Immunocytochemistry,”Academic Press, Orlando, Fla. Vol. 1(1982), Vol. 2 (1983), Vol. 3(1985); Tijssen, “Practice and Theory of Enzyme Immunoassays: LaboratoryTechniques in Biochemistry and Molecular Biology,” Elsevier SciencePublishers, Amsterdam, The Netherlands (1985).

The immunological assay test samples of the present invention includecells, protein or membrane extracts of cells, or biological fluids suchas blood, serum, plasma, or urine. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing protein extracts or membraneextracts of cells are well known in the art and can be readily adaptedin order to obtain a sample which is compatible with the systemutilized.

A kit contains all the necessary reagents to carry out the previouslydescribed methods of detection. The kit may comprise: (i) a firstcontainer containing an above-described antibody, and (ii) secondcontainer containing a conjugate comprising a binding partner of theantibody and a label. In another preferred embodiment, the kit furthercomprises one or more other containers comprising one or more of thefollowing: wash reagents and reagents capable of detecting the presenceof bound antibodies.

Examples of detection reagents include, but are not limited to, labeledsecondary antibodies, or in the alternative, if the primary antibody islabeled, the chromophoric, enzymatic, or antibody binding reagents whichare capable of reacting with the labeled antibody. The compartmentalizedkit may be as described above for nucleic acid probe kits. One skilledin the art will recognize that the antibodies described in the presentinvention can readily be incorporated into one of the established kitformats which are well known in the art.

VIII. Isolation of Natural Binding Partners of PTP05 or PTP10

The present invention also relates to methods of detecting naturalbinding partners capable of binding to a PTP05 polypeptide or to a PTP10polypeptide. A natural binding partner of PTP05 or PTP10 may be, forexample, a substrate protein which is dephosphorylated as part of asignaling cascade. The binding partner(s) may be present within acomplex mixture, for example, serum, body fluids, or cell extracts.

In general, methods for identifying natural binding partners compriseincubating a substance with a first polypeptide, PTP05 or PTP10 for theinvention described herein, and detecting the presence of a substancebound to the first polypeptide. Preferred methods include the two-hybridsystem of Fields and Song (supra) and co-immunoprecipitation wherein thefirst polypeptide is allowed to bind to a natural binding partner, thenthe polypeptide complex is immunoprecipitated using antibodies specificfor the first polypeptide. The natural binding partner can then beisolated and identified by techniques well known in the art.

IX. Identification of and Uses for Substances Capable of ModulatingPTP05 or PTP10 Activity

The present invention also relates to a method of detecting a substancecapable of modulating PTP05 or PTP10 activity. Such substances caneither enhance activity (agonists) or inhibit activity (antagonists).Agonists and antagonists can be peptides, antibodies, products fromnatural sources such as fungal or plant extracts or small molecularweight organic compounds. In general, small molecular weight organiccompounds are preferred. Examples of classes of compounds that can betested for PTP05 or PTP10 modulating activity are, for example but notlimited to, thiazoles (see for example co-pending U.S. applications60/033,522 filed Dec. 19, 1996, Ser. No. 08/660,900 filed Jun. 7, 1996),and naphthopyrones (U.S. Pat. No. 5,602,171, issued Feb. 11, 1997).

In general the method comprises incubating cells that produce PTP05 orPTP10 in the presence of a test substance and detecting changes in thelevel of PTP05 or PTP10 activity or PTP05 or PTP10 binding partneractivity. A change in activity may be manifested by increased ordecreased phosphorylation of a PTP05 or PTP10 polypeptide, increased ordecreased phosphorylation of a PTP05 or PTP10 substrate, increased ordecreased binding to a PTP05 or PTP10 natural binding partner orincreased or decreased biological response in cells. A method fordetecting modulation of PTP05 or PTP10 activity using thephosphorylation of an artificial substrate is shown in the examplesbelow. Biological responses can include, for example, proliferation,differentiation, survival, or motility. The substance thus identifiedwould produce a change in activity indicative of the agonist orantagonist nature of the substance. Once the substance is identified itcan be isolated using techniques well known in the art, if not alreadyavailable in a purified form.

The present invention also encompasses a method of agonizing(stimulating) or antagonizing PTP05 or PTP10 associated activity in amammal comprising administering to said mammal an agonist or antagonistto PTP05 or PTP10 in an amount sufficient to effect said agonism orantagonism. Also encompassed in the present application is a method oftreating diseases in a mammal with an agonist or antagonist of PTP05- orPTP10-related activity comprising administering the agonist orantagonist to a mammal in an amount sufficient to agonize or antagonizePTP05 or PTP10 associated function(s). The particular compound can beadministered to a patient either by itself or in a pharmaceuticalcomposition where it is mixed with suitable carriers or excipient(s). Intreating a patient, a therapeutically effective dose of the compound isadministered. A therapeutically effective dose refers to that amount ofthe compound that results in amelioration of symptoms or a prolongationof survival in a patient.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals. Cell culture assays and animal studies can be used fordetermining the LD₅₀ (the dose lethal to 50% of a population) and theED₅₀ (the dose therapeutically effective in 50% of a population).

The dose ratio between toxic and therapeutic effects is the therapeuticindex, which can be expressed as the ratio LD₅₀/ED₅₀. Compounds whichexhibit large therapeutic indices are preferred. The data obtained fromthese cell culture assays and animal studies can be used in formulatinga range of dosages for use in human. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays by determining an IC₅₀ (i.e., the concentration of thetest compound which achieves a half-maximal disruption of the proteincomplex, or a half-maximal inhibition of the cellular level and/oractivity of a cellular component, ex. PTP05). A dose can then beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans. Levels in plasma may be measured, for example,by HPLC. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g. Fingl et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p1).

It should be noted that the attending physician would know how to andwhen to terminate, interrupt, or adjust administration due to toxicity,or to organ dysfunctions. Conversely, the attending physician would alsoknow to adjust treatment to higher levels if the clinical response werenot adequate (precluding toxicity). The magnitude of an administrateddose in the management of the oncogenic disorder of interest will varywith the severity of the condition to be treated and to the route ofadministration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight, and response of the individual patient. A programcomparable to that discussed above may be used in veterinary medicine.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in “Remington'sPharmaceutical Sciences,” 1990, 18th ed., Mack Publishing Co., Easton,Pa. Suitable routes may include oral, rectal, transdermal, vaginal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections, just to name afew.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Forsuch transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

Use of pharmaceutically acceptable carriers to formulate the compoundsherein disclosed for the practice of the invention into dosages suitablefor systemic administration is within the scope of the invention. Withproper choice of carrier and suitable manufacturing practice, thecompositions of the present invention, in particular those formulated assolutions, may be administered parenterally, such as by intravenousinjection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient to be treated. Particular formulations suitable for parenteraladministration of hydrophobic compounds can be found in U.S. Pat. No.5,610,173, issued Mar. 11, 1997 and U.S. Provisional Application Ser.No. 60/039,870, filed Mar. 5, 1997, both of which are incorporated byreference herein in their entirety.

Agents intended to be administered intra cellularly may be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents may be encapsulated into liposomes, thenadministered as described above. Liposomes are spherical lipid bilayerswith aqueous interiors. All molecules present in an aqueous solution atthe time of liposome formation are incorporated into the aqueousinterior. The liposomal contents are both protected from the externalmicroenvironment and, because liposomes fuse with cell membranes, areefficiently delivered into the cell cytoplasm. Small organic moleculesmay be directly administered intra cellularly due to theirhydrophobicity.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in anamount effective to achieve its intended purpose. Determination of aneffective amount is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

X. Transgenic Animals

Also contemplated by the invention are transgenic animals useful for thestudy of PTP05 or PTP10 activity in complex in vivo systems. A“transgenic animal” is an animal having cells that contain DNA which hasbeen artificially inserted into a cell, which DNA becomes part of thegenome of the animal which develops from that cell. Preferred transgenicanimals are primates, mice, rats, cows, pigs, horses, goats, sheep, dogsand cats. The transgenic DNA may encode for a human PTP05 or PTP10polypeptide. Native expression in an animal may alternatively be reducedby providing an amount of antisense RNA or DNA effective to reduceexpression of the target gene.

A variety of methods are available for the production of transgenicanimals associated with this invention. DNA sequences encoding PTP05 orPTP10 can be injected into the pronucleus of a fertilized egg beforefusion of the male and female pronuclei, or injected into the nucleus ofan embryonic cell (e.g., the nucleus of a two-cell embryo) following theinitiation of cell division (Brinster, et al., Proc. Nat. Acad. Sci. USA82:4438, 1985). Embryos can be infected with viruses, especiallyretroviruses, modified to carry inorganic-ion receptor nucleotidesequences of the invention.

Pluripotent stem cells derived from the inner cell mass of the embryoand stabilized in culture can be manipulated in culture to incorporatenucleotide sequences of the invention. A transgenic animal can beproduced from such cells through implantation into a blastocyst that isimplanted into a foster mother and allowed to come to term. Animalssuitable for transgenic experiments can be obtained from standardcommercial sources such as Charles River (Wilmington, Mass.), Taconic(Germantown, N.Y.), Harlan Sprague Dawley (Indianapolis, Ind.), etc.

The procedures for manipulation of the rodent embryo and for microinjection of DNA into the pronucleus of the zygote are well known tothose of ordinary skill in the art (Hogan, et al., supra). Microinjection procedures for fish, amphibian eggs and birds are detailed inHoudebine and Chourrout, Experientia 47: 897-905, 1991). Otherprocedures for introduction of DNA into tissues of animals are describedin U.S. Pat. No. 4,945,050 (Sandford et al., Jul. 30, 1990).

By way of example only, to prepare a transgenic mouse, female mice areinduced to superovulate. After being allowed to mate, the females aresacrificed by CO₂ asphyxiation or cervical dislocation and embryos arerecovered from excised oviducts. Surrounding cumulus cells are removed.Pronuclear embryos are then washed and stored until the time ofinjection. Randomly cycling adult female mice are paired withvasectomized males. Recipient females are mated at the same time asdonor females. Embryos then are transferred surgically. The procedurefor generating transgenic rats is similar to that of mice. See Hammer,et al., Cell 63:1099-1112, 1990).

Methods for the culturing of embryonic stem (ES) cells and thesubsequent production of transgenic animals by the introduction of DNAinto ES cells using methods such as electroporation, calciumphosphate/DNA precipitation and direct injection also are well known tothose of ordinary skill in the art. (See, for example, Teratocarcinomasand Embryonic Stem Cells, A Practical Approach, E. J. Robertson, ed.,IRL Press, 1987).

In cases involving random gene integration, a clone containing thesequence(s) of the invention is co-transfected with a gene encodingresistance. Alternatively, the gene encoding neomycin resistance isphysically linked to the sequence(s) of the invention. Transfection andisolation of desired clones are carried out by any one of severalmethods well known to those of ordinary skill in the art (E. J.Robertson, supra). DNA molecules introduced into ES cells can also beintegrated into the chromosome through the process of homologousrecombination. (See Capecchi, Science 244: 1288, 1989.) Methods forpositive selection of the recombination event (i.e., neo resistance) anddual positive-negative selection (i.e., neo resistance and gancyclovirresistance) and the subsequent identification of the desired clones byPCR have been described by Capecchi, supra and Joyner et al., Nature338: 153, 1989), the teachings of which are incorporated by referenceherein. The final phase of the procedure is to inject targeted ES cellsinto blastocysts and to transfer the blastocysts into pseudopregnantfemales. The resulting chimeric animals are bred and the offspring areanalyzed by Southern blotting to identify individuals that carry thetransgene. Procedures for the production of non-rodent mammals and otheranimals have been discussed by others. (See Houdebine and Chourrout,supra; Pursel, et al., Science 244:1281, 1989; Simms, et al.,Bio/Technology 6:179, 1988.)

Thus, the invention provides transgenic, nonhuman mammals containing atransgene encoding a PTP05 polypeptide or a PTP10 polypeptide or a geneeffecting the expression of a PTP05 polypeptide or a PTP10 polypeptide.Such transgenic nonhuman mammals are particularly useful as an in vivotest system for studying the effects of introducing a PTP05 polypeptideor a PTP10 polypeptide, or for regulating the expression of a PTP05polypeptide or a PTP10 polypeptide (i.e., through the introduction ofadditional genes, antisense nucleic acids, or ribozymes).

XI. Gene Therapy

PTP05 and/or PTP10 nucleic acid sequences, both mutated and non-mutated,will also be useful in gene therapy (reviewed in Miller, Nature357:455-460, (1992). Miller states that advances have resulted inpractical approaches to human gene therapy that have demonstratedpositive initial results. The basic science of gene therapy is describedin Mulligan, Science 260:926, 1993). As used herein “gene therapy” is aform of gene transfer and is included within the definition of genetransfer as used herein and specifically refers to gene transfer toexpress a therapeutic product from a cell in vivo or in vitro. Genetransfer can be performed ex vivo on cells which are then transplantedinto a patient, or can be performed by direct administration of thenucleic acid or nucleic acid-protein complex into the patient.

In one preferred embodiment, an expression vector containing a PTP05coding sequence, a PTP05 mutant coding sequence, a PTP10 coding sequenceor a PTP10 mutant coding sequence, as described above, is inserted intocells, the cells are grown in vitro and then infused in large numbersinto patients. In another preferred embodiment, a DNA segment containinga promoter of choice (for example a strong promoter) is transferred intocells containing an endogenous PTP05 or PTP10 in such a manner that thepromoter segment enhances expression of the endogenous PTP05 gene (orPTP10) (for example, the promoter segment is transferred to the cellsuch that it becomes directly linked to the endogenous PTP gene).

The gene therapy may involve the use of an adenovirus containing PTP05or PTP10 cDNA targeted to an appropriate cell type, systemic PTP05 orPTP10 increase by implantation of engineered cells, injection with PTP05or PTP10 virus, or injection of naked PTP05 or PTP10 DNA intoappropriate cells or tissues, for example adipose tissue.

Expression vectors derived from viruses such as retroviruses, vacciniavirus, adenovirus, adeno-associated virus, herpes viruses, other RNAviruses, or bovine papilloma virus, may be used for delivery ofnucleotide sequences (e.g., cDNA) encoding recombinant PTP05 or PTP10protein into the targeted cell population (e.g., tumor cells or fatcells). Methods which are well known to those skilled in the art can beused to construct recombinant viral vectors containing coding sequences.See, for example, the techniques described in Maniatis et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.(1989), and in Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates and Wiley Interscience, N.Y. (1989).Alternatively, recombinant nucleic acid molecules encoding proteinsequences can be used as naked DNA or in reconstituted system e.g.,liposomes or other lipid systems for delivery to target cells (See e.g.,Felgner et al., Nature 337:387-8, 1989). Several other methods for thedirect transfer of plasmid DNA into cells exist for use in human genetherapy and involve targeting the DNA to receptors on cells bycomplexing the plasmid DNA to proteins. See, Miller, supra.

In its simplest form, gene transfer can be performed by simply injectingminute amounts of DNA into the nucleus of a cell, through a process ofmicro injection. (Capecchi M R, Cell 22:479-88, 1980). Once recombinantgenes are introduced into a cell, they can be recognized by the cell'snormal mechanisms for transcription and translation, and a gene productwill be expressed. Other methods have also been attempted forintroducing DNA into larger numbers of cells. These methods include:transfection, wherein DNA is precipitated with CaPO₄ and taken intocells by pinocytosis (Chen C. and Okayama H, Mol. Cell Biol. 7:2745-52,1987); electroporation, wherein cells are exposed to large voltagepulses to introduce holes into the membrane (Chu G., et al., NucleicAcids Res., 15:1311-26, 1987); lipofection/liposome fusion, wherein DNAis packaged into lipophilic vesicles which fuse with a target cell(Felgner P L., et al., Proc. Natl. Acad. Sci. USA. 84:7413-7, 1987); andparticle bombardment using DNA bound to small projectiles (Yang N S. etal., Proc. Natl. Acad. Sci. 87:9568-72, 1990). Another method forintroducing DNA into cells is to couple the DNA to chemically modifiedproteins.

It has also been shown that adenovirus proteins are capable ofdestabilizing endosomes and enhancing the uptake of DNA into cells. Theadmixture of adenovirus to solutions containing DNA complexes, or thebinding of DNA to polylysine covalently attached to adenovirus usingprotein crosslinking agents substantially improves the uptake andexpression of the recombinant gene. (Curiel, et al., Am. J. Respir.Cell. Mol. Biol., 6:247-52, 1992).

As used herein “gene transfer” means the process of introducing aforeign nucleic acid molecule into a cell. Gene transfer is commonlyperformed to enable the expression of a particular product encoded bythe gene. The product may include a protein, polypeptide, antisense DNAor RNA, or enzymatically active RNA. Gene transfer can be performed incultured cells or by direct administration into animals. Generally genetransfer involves the process of nucleic acid contact with a target cellby non-specific or receptor mediated interactions, uptake of nucleicacid into the cell through the membrane or by endocytosis, and releaseof nucleic acid into the cytoplasm from the plasma membrane or endosome.Expression may require, in addition, movement of the nucleic acid intothe nucleus of the cell and binding to appropriate nuclear factors fortranscription.

In another preferred embodiment, a vector having nucleic acid sequencesencoding a PTP05 or PTP10 is provided in which the nucleic acid sequenceis expressed only in specific tissue. Methods of achievingtissue-specific gene expression as set forth in InternationalPublication No. WO 93/09236, filed Nov. 3, 1992 and published May 13,1993.

In all of the preceding vectors set forth above, a further aspect of theinvention is that the nucleic acid sequence contained in the vector mayinclude additions, deletions or modifications to some or all of thesequence of the nucleic acid, as defined above.

In another preferred embodiment, a PTP05 or PTP10 nucleic acid is usedin gene replacement. “Gene replacement” as used herein means supplying anucleic acid sequence which is capable of being expressed in vivo in ananimal and thereby providing or augmenting the function of an endogenousgene which is missing or defective in the animal. Methods of introducingthe nucleic acid into the animal to be treated are as described above.

XII. Compounds that Modulate the Function of PTP05 or PTP10 Proteins

In an effort to discover novel treatments for diseases, biomedicalresearchers and chemists have designed, synthesized, and testedmolecules that inhibit the function of protein kinases. Some smallorganic molecules form a class of compounds that modulate the functionof protein kinases. Examples of molecules that have been reported toinhibit the function of protein kinases include, but are not limited to,bis monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO92/20642, published Nov. 26, 1992 by Maguire et al.), vinylene-azaindolederivatives (PCT WO 94/14808, published Jul. 7, 1994 by Ballinari etal.), 1-cyclopropyl-4-pyridyl-quinolones (U.S. Pat. No. 5,330,992),styryl compounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridylcompounds (U.S. Pat. No. 5,302,606), certain quinazoline derivatives (EPApplication No. 0 566 266 A1), seleoindoles and selenides (PCT WO94/03427, published Feb. 17, 1994 by Denny et al.), tricyclicpolyhydroxylic compounds (PCT WO 92/21660, published Dec. 10, 1992 byDow), and benzylphosphonic acid compounds (PCT WO 91/15495, publishedOct. 17, 1991 by Dow et al). The compounds that can traverse cellmembranes and are resistant to acid hydrolysis are potentiallyadvantageous therapeutics as they can become highly bioavailable afterbeing administered orally to patients. However, many of these proteinkinase inhibitors only weakly inhibit the function of protein kinases.In addition, many inhibit a variety of protein kinases and willtherefore cause multiple side-effects as therapeutics for diseases.

Some indolinone compounds, however, form classes of acid resistant andmembrane permeable organic molecules. WO 96/22976, published Aug. 1,1996 by Ballinari et al. describes hydrosoluble indolinone compoundsthat harbor tetralin, naphthalene, quinoline, and indole substituentsfused to the oxindole ring. These bicyclic substituents are in turnsubstituted with polar moieties including hydroxylated alkyl, phosphate,and ether moieties. U.S. patent application Ser. No. 08/702,232, filedAug. 23, 1996, entitled “Indolinone Combinatorial Libraries and RelatedProducts and Methods for the Treatment of Disease” by Tang et al. (Lyon& Lyon Docket No. 221/187) and 08/485,323, filed Jun. 7, 1995, entitled“Benzylidene-Z-Indoline Compounds for the Treatment of Disease” by Tanget al. (Lyon & Lyon Docket No. 223/298) and International PatentPublication WO 96/22976, published Aug. 1, 1996 by Ballinari et al., allof which are incorporated herein by reference in their entirety,including any drawings, describe indolinone chemical libraries ofindolinone compounds harboring other bicyclic moieties as well asmonocyclic moieties fused to the oxindole ring. Application Ser. No.08/702,232, filed Aug. 23, 1996, entitled “Indolinone CombinatorialLibraries and Related Products and Methods for the Treatment of Disease”by Tang et al. (Lyon & Lyon Docket No. 221/187), 08/485,323, filed Jun.7, 1995, entitled “Benzylidene-Z-Indoline Compounds for the Treatment ofDisease” by Tang et al. (Lyon & Lyon Docket No. 223/298), and WO96/22976, published Aug. 1, 1996 by Ballinari et al. teach methods ofindolinone synthesis, methods of testing the biological activity ofindolinone compounds in cells, and inhibition patterns of indolinonederivatives.

Other examples of substances capable of modulating PTP05 or PTP10activity include, but are not limited to, tyrphostins, quinazolines,quinoxolines, and quinolines.

The quinazolines, tyrphostins, quinolines, and quinoxolines referred toabove include well known compounds such as those described in theliterature. For example, representative publications describingquinazoline include Barker et al., EPO Publication No. 0 520 722 A1;Jones et al., U.S. Pat. No. 4,447,608; Kabbe et al., U.S. Pat. No.4,757,072; Kaul and Vougioukas, U.S. Pat. No. 5,316,553; Kreighbaum andComer, U.S. Pat. No. 4,343,940; Pegg and Wardleworth, EPO PublicationNo. 0 562 734 A1; Barker et al., Proc. of Am. Assoc. for Cancer Research32:327 (1991); Bertino, J. R., Cancer Research 3:293-304 (1979);Bertino, J. R., Cancer Research 9(2 part 1):293-304 (1979); Curtin etal., Br. J. Cancer 53:361-368 (1986); Fernandes et al., Cancer Research43:1117-1123 (1983); Ferris et al. J. Org. Chem. 44 (2):173-178; Fry etal., Science 265:1093-1095 (1994); Jackman et al., Cancer Research51:5579-5586 (1981); Jones et al. J. Med. Chem. 29(6):1114-1118; Lee andSkibo, Biochemistry 26(23):7355-7362 (1987); Lemus et al., J. Org. Chem.54:3511-3518 (1989); Ley and Seng, Synthesis 1975:415-522 (1975);Maxwell et al., Magnetic Resonance in Medicine 17:189-196 (1991); Miniet al., Cancer Research 45:325-330 (1985); Phillips and Castle, J.Heterocyclic Chem. 17(19):1489-1596 (1980); Reece et al., CancerResearch 47(11):2996-2999 (1977); Sculier et al., Cancer Immunol. andImmunother. 23:A65 (1986); Sikora et al., Cancer Letters 23:289-295(1984); Sikora et al., Analytical Biochem. 172:344-355 (1988); all ofwhich are incorporated herein by reference in their entirety, includingany drawings.

Quinoxaline is described in Kaul and Vougioukas, U.S. Pat. No.5,316,553, incorporated herein by reference in its entirety, includingany drawings.

Quinolines are described in Dolle et al., J. Med. Chem. 37:2627-2629(1994); MaGuire, J. Med. Chem. 37:2129-2131 (1994); Burke et al., J.Med. Chem. 36:425-432 (1993); and Burke et al. BioOrganic Med. Chem.Letters 2:1771-1774 (1992), all of which are incorporated by referencein their entirety, including any drawings.

Tyrphostins are described in Allen et al., Clin. Exp. Immunol.91:141-156 (1993); Anafi et al., Blood 82:12:3524-3529 (1993); Baker etal., J. Cell Sci. 102:543-555 (1992); Bilder et al., Amer. Physiol. Soc.pp. 6363-6143:C721-C730 (1991); Brunton et al., Proceedings of Amer.Assoc. Cancer Rsch. 33:558 (1992); Bryckaert et al., Experimental CellResearch 199:255-261 (1992); Dong et al., J. Leukocyte Biology 53:53-60(1993); Dong et al., J. Immunol. 151(5):2717-2724 (1993); Gazit et al.,J. Med. Chem. 32:2344-2352 (1989); Gazit et al., “J. Med. Chem.36:3556-3564 (1993); Kaur et al., Anti-Cancer Drugs 5:213-222 (1994);Kaur et al., King et al., Biochem. J. 275:413-418 (1991); Kuo et al.,Cancer Letters 74:197-202 (1993); Levitzki, A., The FASEB J. 6:3275-3282(1992); Lyall et al., J. Biol. Chem. 264:14503-14509 (1989); Peterson etal., The Prostate 22:335-345 (1993); Pillemer et al., Int. J. Cancer50:80-85 (1992); Posner et al., Molecular Pharmacology 45:673-683(1993); Rendu et al., Biol. Pharmacology 44(5):881-888 (1992); Sauro andThomas, Life Sciences 53:371-376 (1993); Sauro and Thomas, J. Pharm. andExperimental Therapeutics 267(3):119-1125 (1993); Wolbring et al., J.Biol. Chem. 269(36):22470-22472 (1994); and Yoneda et al., CancerResearch 51:4430-4435 (1991); all of which are incorporated herein byreference in their entirety, including any drawings.

Other compounds that could be used as modulators include oxindolinonessuch as those described in U.S. patent application Ser. No. 08/702,232filed Aug. 23, 1996, incorporated herein by reference in its entirety,including any drawings.

EXAMPLES Example 1 Isolation of cDNA Clones Encoding PTP05 and PTP10

The example below describes the isolation and identification of new PTPsequences from primary murine fat and rat basal forebrain and thesubsequent cloning of a full-length PTP05 sequence Also described areprobes useful for the detection of PTP05 and/or PTP10 in cells ortissues.

Materials and Methods:

Total RNAs were isolated using the Guanidine Salts/Phenol extractionprotocol of Chomczynski and Sacchi (P. Chomczynski and N. Sacchi, Anal.Biochem. 162, 156 (1987) from ob/ob mouse fat and, separately, rat basalforebrain. These RNAs were used to generate single-stranded cDNA usingthe Superscript Preamplification System (GIBCO BRL, Gaithersburg, Md.;Gerard, et al, FOCUS 11:66, 1989) under conditions recommended by themanufacturer. A typical reaction used 10 μg total RNA with 1.5 μgoligo(dT)₁₂₋₁₈ in a reaction volume of 60 μL. The product was treatedwith RNaseH and diluted to 100 μL with H₂0. For subsequent PCRamplification, 1-4 μL of this sscDNA was used in each reaction.

Degenerate oligonucleotides were synthesized on an Applied Biosystems394 DNA synthesizer using established phosphoramidite chemistry,precipitated with ethanol and used unpurified for PCR. The sequence ofthe degenerate oligonucleotide primers follows;

-   -   PTPDFW=5′-GAYTTYTGGVRNATGRTNTGGGA-(sense) (SEQ ID NO:9) and    -   PTPHCSA=5′-CGGCCSAYNCCNGCNSWRCARTG-3′ (antisense) (SEQ ID        NO:10).

These primers were derived from the peptide sequences DFWXMXW(E/D) (SEQID NO:11) (sense strand from PTP catalytic domain) and HCXAGXG (SEQ IDNO:12) (antisense strand from PTP catalytic domain), respectively. Thestandard UIPAC designations for degenerate residue designations are:N=A, C, G, or T; R=A or G; Y=C or T; V=A, C or G; W=C or T; S=C or G;M=A or C; and H=A, C or T.

PCR reactions were performed using degenerate primers applied to thesingle-stranded cDNA listed above. The primers were added at a finalconcentration of 5 μM each to a mixture containing 10 mM Tris.HCl (pH8.3), 50 mM KCl, 1.5 mM MgCl₂, 200 μM each deoxynucleoside triphosphate,0.001% gelatin, 1.5 U AmpliTaq DNA Polymerase (Perkin-Elmer/Cetus), and1-4 μL cDNA. Following 3 min denaturation at 95° C., the cyclingconditions were 94° C. for 30 sec, 50° C. for 1 min, and 72° C. for 1min 45 sec for 35 cycles. PCR fragments migrating between 350-400 bpwere isolated from 2% agarose gels using the GeneClean Kit (Bio101), andT-A cloned into the pCRII vector (Invitrogen Corp. U.S.A.) according tothe manufacturer's protocol.

Colonies were selected for mini-plasmid DNA-preparations using Qiagencolumns and the plasmid DNA was sequenced using cycle sequencingdye-terminator kit with AmpliTaq DNA Polymerase, FS (ABI, Foster City,Calif.). Sequencing reaction products were run on an ABI Prism 377 DNASequencer, and analyzed using the BLAST alignment algorithm (Altschul,S. F. et al., J. Mol. Biol. 215:403-10). Several copies of a cloneencoding a novel PTP (R90-2-22), designated SuPTP05, was isolated frommurine adipose tissue. A related clone, PTP10, was isolated from ratbasal forebrain.

To obtain full-length cDNA encoding the novel phosphatase PTP05, RACE(rapid amplification of cDNA ends) was performed with sense oranti-sense oligonucleoides derived from the original PCR fragments.Marathon-Ready cDNA (Clontech, Palo Alto, Calif.) made from mouse testiswas used in the RACE reactions with the following primers: RACE primers:(SEQ ID NO:13) 5′-CACCGTTCGAGTATTTCAGATTGTGAAGAAGTCC-3′ (6595), (SEQ IDNO:14) 5′-GGACTTCTTCACAATCTGAAATACTCGAACGGTG-3′ (6596), (SEQ ID NO:15)5′-CCGTTATGTGAGGAAGAGCCACATTACAGGACC-3′ (6599), (SEQ ID NO:16)5′-GGTCCTGTAATGTGGCTCTTCCTCACATAACGG-3′ (6600), AP-1, and AP-2(Clontech). RT-PCR primers for PTP05 sequeqncing: (SEQ ID NO:17)5′-CACCGTTCGAGTATTTCAGATTGTGAAGAAGTCC-3′ (6595), (SEQ ID NO:18)5′-GGTCCTGTAATGTGGCTCTTCCTCACATAACGG-3′ (6600).

Isolated cDNA fragments encoding SuPTP05 were confirmed by DNAsequencing and subsequently used as probes for the screening of a murinetestis cDNA library.

Two murine testis cDNA libraries (lZapII, Stratagene, La Jolla, Calif.and lgt10, Clontech), were screened to isolate full-length transcriptsencoding PTP05. The 5′ or 3′-RACE fragments were ³²P-labeled by randompriming and used as hybridization probes at 2×10⁶ cpm/mL followingstandard techniques for library screening. Pre-hybridization (3 hrs) andhybridization (overnight) were conducted at 42° C. in 5×SSC, 5×Denhart'ssolution, 2.5% dextran sulfate, 50 mM Na₂PO₄/NaHPO4 [pH 7.0], 50%formamide with 100 mg/mL denatured salmon sperm DNA. Stringent washeswere performed at 65° C. in 0.1×SSC and 0.1% SDS. Several overlappingclones were isolated and found to span the collective sequences of thePCR fragment (R90-2-22) and the RACE products. The final sequence wasverified by sequencing of both strains using a cycle sequencingdye-terminator kit with AmpliTaq DNA Polymerase, FS (ABI, Foster City,Calif.). Sequencing reaction products were run on an ABI Prism 377 DNASequencer. A full-length PTP10 clone can be obtained using the sametechniques.

Results:

The primary murine PTP05 transcript is 1785 nucleotides and encodes apredicted polypeptide of 426 amino acids with a predicted molecularweight of 49122 daltons (SEQ ID NO:1 and SEQ ID NO:5). The PTP05 codingsequence is flanked by a 198 nucleotide 5′-untranslated region and a 279nucleotide 3′-untranslated region ending with a poly(A) tail. There areinframe stop codons in all three frames upstream of the primary openreading frame. The ATG beginning at nucleotide position 199 conforms tothe Kozak consensus for an initiating methionine. One clone (#6.1)contains an insertion of 111 bp at nucleotide 328 resulting in anaddition 37 amino acids added inframe to the coding sequence. A secondclone (#10.1) has a deletion of 93 bp beginning at nucleotide 1415,resulting in a frame-shift and premature termination. Upstream of the198 bp 5′UTR, the numerous clones diverge into 2 groups, extending the5′UTR an additional 98-153 bp. Furthermore, one clone (#15.3) lacks thepolyA tail at nucleotide 1758 extends the 3′ UTR by another 300nucleotides.

The amino acid sequence shows no signal sequence or a transmembranedomain, and PTP05 is therefore predicted to be an intracellular protein.The N-terminal domain of murine PTP05 extends from amino acid 1 to 187and is unique, ie. contains no significant homology to any protein inthe non-redundant protein database. The non-redundant protein databaseconsists of peptide sequences from GenBank Genpept, PIR, and SwissProt.There is a single protein tyrosine phosphatase catalytic domainextending from amino acids 188-420. The catalytic domain shares arelatively low level of identity at the amino acid level (40-47%) toPTPs from 5 distinct families: ZPEP (mouse) (46.7%), PTP-BAS (human)(45.6%), DEP (human) (40.5%), PTP-g (human) (40.6%), suggesting that itrepresents a new family of PTPs. The C-terminal tail of PTP05 extendsbeyond the catalytic domain from amino acids 421-426 and is nothomologous to other protein tyrosine phosphatases. Motifs found in thecytoplasmic domain of other mammalian PTPs that are absent from PTP05include: SH2, Talin/Ezrin-like, PEST, GLGF, and Retinaldehyde-bindingprotein domains. Owing to its divergent catalytic domain and absence ofwell-known non-catalytic motifs, we have designated PTP05 as a new anddistinct family of protein tyrosine phosphatases.

An alternative form of murine PTP05 contains an insertion of 111-bp inthe N-terminal coding region, extending the sequence by 37 a (SEQ IDNO:2 and SEQ ID NO:6). This 1,896 bp “long” form of murine PTP05 encodesa polypeptide of 463 amino acids with a predicted molecular weight of53716 daltons. The insertion is located at amino acid positions 44-80and is not significantly homologous to other proteins in thenon-redundant protein database.

A third form of PTP05 has a deletion of nucleotides 1415-1507 resultingin a frame shift and C-terminal truncation leading to an alternatesequence from amino acids 406-412 (SEQ ID NO:3 and SEQ ID NO:7). The1,692 bp “C-trunc” murine PTP05 encodes a polypeptide of 412 amino acidswith a predicted molecular weight of 47233 daltons.

The rat PTP10 clone shares 92% identity at the DNA level (320nucleotides) and 85% amino acid identity at the protein level (107 aminoacids) with murine PTP05 (See FIG. 1). The level of homology of the twocatalytic domains suggests that PTP05 and PTP10 are distinct but relatedgenes, and thus PTP10 is considered to be a second member of this newPTP family. Partial sequences of rat PTP10 are shown in SEQ ID NO:4(nucleic acid) and SEQ ID NO:8 (amino acid).

Example 2 Expression of PTP05

The example below shows the evaluation of PTP05 and PtP10 expression innormal murine tissues. A similar analysis can be done in human tissuesusing a human PTP05 or PTP10.

Materials and Methods:

A mouse normal tissue Northern blot containing 2 μg polyA+ mRNA per lanefrom 8 different mouse adult tissues (lung, testis, brain, heart, liver,kidney, spleen, skeletal muscle) on a charge-modified nylon membrane wasobtained from Clontech (#7762-1, Palo Alto, Calif.).

The membrane was hybridized with randomly primed [a³²P]dCTP-labeledprobe synthesized from a 241 bp EcoRI fragment of R90-2-22 (see above).Hybridization was performed at 42° C. overnight in 5×SSC, 2% SDS,10×Denhardt's solution, 50% formamide, 100 μg/mL denatured salmon spermDNA with 1-2×10⁶ cpm/mL of ³²P-labeled DNA probe. The membrane waswashed at room temperature in 2×SSC/0.05% SDS for 30 min and followed by50° C. in 0.2×SSC/0.1% SDS for 30 min, and exposed overnight on KodakXAR-2 film.

A similar analysis was performed using the 320 bp rat PTP10 fragment asa probe of a rat normal tissue Norther blot.

RT-PCR Detection of Novel PTPs

Total RNA was isolated from fresh frozen mouse or rat (separately)tissues by centrifugation through a cesium chloride cushion. Twenty μgof total RNA was reverse transcribed with random hexamers and Moloneymurine leukemia virus reverse transcriptase (Super-ScriptII, GIBCO BRL,Gaithersburg, Md.). PCR was then used to amplify cDNA encoding SuPTP05.RT-PCR reactions lacking only the reverse transcriptase were performedas controls. PCR products were electrophoresed on 3% agarose gels,visualized by ethidium bromide staining and photographed on a UV lightbox. The intensity for a 161-bp fragment specific to murine PTP05 werecompared among different RNA samples. A rating of 3 represents largequantities of PTP05 transcript identified by Northern blot analysiswhile a rating of 0 represents little or none of the transcript wasdetected.

Results:

By Northern analysis, a single murine PTP05 mRNA transcript ofapproximately 3.4 kb was identified, and found to be exclusivelyexpressed in the testis. The lung, brain, heart, liver, kidney, spleen,skeletal muscle samples were negative. PTP10 hybridized to a slightlysmaller band and was also found only in the testis in this analysis.Northern analysis identified two rat PTP10 mRNA transcripts ofapproximately 3.3 kb and 1.8 kb, exclusively expressed in the testis.The rat heart, brain, spleen, lung, liver, skeletal muscle, and kidneysamples were negative.

RT-PCR with gene specific primer-pairs showed that expression of thetranscripts encoding PTP05 confirmed the results from Northern analysisand also detected low levels in adipose, kidney, small intestine, andcells/tissues of hematopoietic or immune origin including spleen,thymus, lymph node, bone marrow, and peripheral blood lymphocytes).RT-PCR with rat PTP10 gene specific primers confirmed the results fromthe Northern analysis, detecting a strong signal only in rat testissscDNA and not in templates corresponding to rat skeletal muscle, heart,kidney, spleen, adrenal gland, lung, liver, intestine, uterus, spinalcord, brain, cortex and ovary.

The relatively selective expression of PTP05 in cells of hematopoetic orimmune origin suggests a potential involvement in immune regulationincluding T and B cell survival, differentiation or co-stimulation,and/or inflammatory, immunosuppressive or autoimmune disorders.Additionally, expression in adipose tissue (also the source from whichPTP05 was originally isolated) suggests a possible role in metabolicdisorders such as diabetes.

Example 3 Recombinant Expression of PTP05

The following example illustrates the construction of vectors forexpression of recombinant PTP05 and the creation of recombinant celllines expressing PTP05. Similar vectors and recombinant cell lines canbe generated using PTP10 and the techniques described herein.

Construction of Expression Vectors

Expression constructs were generated by PCR-assisted mutagenesis inwhich the entire coding domain of PTP05 was tagged on itscarboxy-terminal end with the hemophilus influenza hemaglutinin (HA)epitope YPYDVPDYAS (SEQ ID NO:19) (Pati, supra). This construct wereintroduced into two mammalian expression vectors: pLXSN (Miller, A. D. &Rosman, G. J., Biotechniques 7, 980-988, 1989) for the generation ofvirus producing lines; and pRK5 for transient expression in mammaliancells.

Dominant negative PTP05 constructs were also made in both pLXSN and pRK5by mutation of the invariant Cys in the conserved His-Cys-Ser-Ala-Glymotif (SEQ ID NO:20) to an Ala by PCR mutagenesis.

The entire PTP05 open reading frame excluding the initiating methionineswas generated by PCR and ligated into pGEX vector for bacterialproduction of GST-fusion proteins for immunization of rabbits forantibody production. This vector contains the glutathione-S-transferasecoding sequence followed by a polylinker for generating recombinantfusion proteins. The GST moiety comprises the N-terminal portion of thefusion protein.

Transient Expression in Mammalian Cells

The pRK5 expression plasmids (10 ug DNA/100 mm plate) containing theHA-tagged PTP05 gene can be introduced into COS and 293 cells withlipofectamine (Gibco BRL). After 72 hours, the cells were harvested in0.5 mL solubilization buffer (20 mM HEPES pH7.35, 150 mM NaCl, 10%glycerol, 1% Triton X-100, 1.5 mM MgCl₂, 1 mM EGTA, 2 mMphenylmethylsulfonyl fluoride, 1 μg/ml aprotinin). Sample aliquots wereresolved by SDS polyacrylamide gel electrophoresis (PAGE) on 15%acrylamide/0.5% bis-acrylamide gels and electrophoretically transferredto nitrocellulose. Non-specific binding was blocked by preincubatingblots in Blotto (phosphate buffered saline containing 5% w/v non-fatdried milk and 0.2% v/v nonidet P-40 (Sigma)), and recombinant proteinwas detected using a murine Mab to the HA decapeptide tag.Alternatively, recombinant protein can be detected using variousPTP05-specific antisera.

Generation of Virus Producing Cell Lines

pLXSN recombinant constructs containing the PTP05 gene were transfectedinto an amphotropic helper cell line PA317 using CaCl₂ mediatedtransfection. After selection on G418, the cells were plated on normalmedia without G418 (500 μg/mL). Supernatants from resistant cells wereused to infect the ecotropic helper cell line GP+E86, and cells againselected on G418. Resistant cells were again taken off G418, and thesupernatants harvested every 8-12 hours and pooled as virus stock.Redemann et al., 1992, Mol. Cell. Biol. 12: 491-498. Viral stock titerswere typically ˜10⁶/mL.

Stable Expression in Mammalian Cells

NIH-3T3, and BALB/3T3 cells were grown in 100 mm plates with DMEM(Gibco) containing 10% fetal calf serum (FCS). The cells weresuperinfected with the PTP05 retrovirus by adding approximately 3 mLviral supernatant to 15 mL culture media for approximately 24 hours.Cells expressing the retroviral constructs were then selected by growthin DMEM/10% FCS supplemented with 500 μg/mL G418.

Example 4 Generation of Anti-PTP05 Antibodies

PTP05-specific immunoreagents were raised in rabbits against a pool ofthree KLH-conjugated synthetic peptides corresponding to uniquesequences present in human PTO05 or PTP10. The peptides (see below) wereconjugated at the C-terminal residue with KLH.

Peptides Used for Immunizing Rabbits: PTP05: (SEQ ID NO:21) peptide433A-MSSPRKVRGKTGRDNDEEEGNSGNLNLRN (SEQ ID NO:22) peptide431A-SPVLSGSSRLSKDTETSVSEKELTQLAQI and (SEQ ID NO:23) peptide432A-WDVSDRSLRNRWNSMDSETAGPSKTVSPV.

Additional immunoreagents were generated by immunizing rabbits with apurified preparation of a GST-fusion protein containing the entirecoding region of PTP05. The GST-fusion proteins were produced inDH5-alpha E. coli bacteria as described in Smith, et al Gene 67:31,1988. Bacterial protein lysates were purified on glutathione-sepharosematrix as described in Smith, et al., supra.

Example 5 Assay for PTP05 Activity

Materials and Methods:

Recombinant wild-type and dominant negative (signaling incompetant)PTP05 (see Example 3, supra) were purified from bacteria as GST-fusionproteins. Lysates were bound to a glutathione-sepharaose matrix andwashed twice with 1×HNTG, followed by one wash with a buffer containing100 mM 2-(N-morpholino)ethansulfonic acid (MES), pH 6.8, 150 mM NaCl,and 1 mM EDTA.

The assay for phosphatase activity was essentially done as described byPei et al. (1993) using p-nitrophenolphosphate (PNPP) as a generic PTPsubstrate. Briefly, after the last washing step, reactions were startedby adding 50 mL Assay Buffer (100 mM MES pH 6.8, 150 mM NaCl, 10 mM DTT,2 mM EDTA, and 50 mM PNPP) to the matrix bound proteins. Samples wereincubated for 20 min. at 23° C. The reactions were terminated by mixing40 μL of each sample with 960 μL 1 N NaOH, and the absorbance ofp-nitrophenol was determined at 450 nm. To control for the presence ofPTP05 in the precipitates, the precipitates were boiled in SDS samplebuffer and analyzed by SDS-PAGE. The presence of PTP05 was then detectedby immunoblot analysis with anti-PTP05 antibodies.

Example 6 Screening Systems for the Identification of Inhibitors ofPTP05 or PTP10 Activity

Assays may be performed in vitro or in vivo and are described in detailherein or can be obtained by modifying existing assays, such as thegrowth assay described in patent application Ser. No. 08/487,088 (Lyon &Lyon Docket No. 212/276), filed Jun. 7, 1995, by Tang et al., andentitled “Novel Pharmaceutical Compounds,” or the assays described inpatent application Ser. No. 60/005,167 (Lyon & Lyon Docket No. 215/256),filed Oct. 13, 1995 by Seedorf et al., and entitled “Diagnosis andTreatment of TKA-1 Related Disorders,” all of which are herebyincorporated herein by reference in their entirety including anydrawings. Another assay which could be modified to use the genes of thepresent invention is described in International Application No. WO94/23039, published Oct. 13, 1994, hereby incorporated herein byreference in its entirety including any drawings. Other possibilitiesinclude detecting kinase activity in an autophosphorylation assay ortesting for kinase activity on standard substrates such as histones,myelin basic protein, gamma tubulin, or centrosomal proteins. Bindingpartners may be identified by putting the N-terminal portion of theprotein into a two-hybrid screen or detecting phosphotyrosine of a dualspecificity kinase (Fields and Song, U.S. Pat. No. 5,283,173, issuedFeb. 1, 1994, incorporated by reference herein, including any drawings).

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Themolecular complexes and the methods, procedures, treatments, molecules,specific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. It will be readily apparentto one skilled in the art that varying substitutions and modificationsmay be made to the invention disclosed herein without departing from thescope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising,” “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group. For example, if X isdescribed as selected from the group consisting of bromine, chlorine,and iodine, claims for X being bromine and claims for X being bromineand chlorine are fully described.

In view of the degeneracy of the genetic code, other combinations ofnucleic acids also encode the claimed peptides and proteins of theinvention. For example, all four nucleic acid sequences GCT, GCC, GCA,and GCG encode the amino acide alanine. Therefore, if for an amino acidthere exists an average of three codons, a polypeptide of 100 aminoacids in length will, on average, be encoded by 3¹⁰⁰, or 5×10⁴⁷ nucleicacid sequences. Thus, a nucleic acid sequence can be modified to form asecond nucleic acid sequence, encoding the same polypeptide as endodedby the first nucleic acid sequences, using routine procedures andwithout undue experimentation. Thus, all possible nucleic acids thatencode the claimed peptides and proteins are also fully describedherein, as if all were written out in full taking into account the codonusage, especially that preferred in humans. Furthermore, changes in theamino acid sequences of polypeptides, or in the corresponding nucleicacid sequence encoding such polypeptide, may be designed or selected totake place in an area of the sequence where the significant activity ofthe polypeptide remains unchanged. For example, an amino acid change maytake place within a β-turn, away from the active site of thepolypeptide. Also changes such as deletions (e.g. removal of a segmentof the polypeptide, or in the corresponding nucleic acid sequenceencoding such polypeptide, which does not affect the active site) andadditions (e.g. addition of more amino acids to the polypeptide sequencewithout affecting the function of the active site, such as the formationof GST-fusion proteins, or additions in the corresponding nucleic acidsequence encoding such polypeptide without affecting the function of theactive site) are also within the scope of the present invention. Suchchanges to the polypeptides can be performed by those with ordinaryskill in the art using routine procedures and without undueexperimentation. Thus, all possible nucleic and/or amino acid sequencesthat can readily be determined not to affect a significant activity ofthe peptide or protein of the invention are also fully described herein.

Other embodiments are within the following claims.

1. An antibody or antibody fragment having specific binding affinity to(i) a PTP05 polypeptide, (ii) a PTP10 polypeptide, (iii) a PTP05 domainpolypeptide, or (iv) a PTP10 domain polypeptide.
 2. The antibody orantibody fragment of claim 1, wherein the PTP05 polypeptide comprises asequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6and SEQ ID NO:7.
 3. The antibody or antibody fragment of claim 1,wherein the PTP10 polypeptide comprises the sequence of SEQ ID NO:8. 4.The antibody or antibody fragment of claim 1, wherein the PTP05 domainor the PTP10 domain is a portion of any one of SEQ ID NOs: 5, 6, 7, or 8that is homologous in sequence to an amino acid sequence from a knownprotein that is predictive of a common function, interaction oractivity.
 5. The antibody or antibody fragment of claim 4, wherein thePTP05 domain or the PTP10 domain is homologous in sequence to the aminoacid sequence of (i) an Src Homology 2 domain, (ii) an Src Homology 3domain, or (iii) a pleckstrin domain.
 6. The antibody or antibodyfragment of claim 4, wherein the PTP05 domain or the PTP10 domain has arelative homology of at least 90% to the amino acid sequence from theknown protein.
 7. The antibody or antibody fragment of claim 6, whereinthe PTP05 domain or the PTP10 domain has a relative homology of at least95% to the amino acid sequence from the known protein.
 8. The antibodyor antibody fragment of claim 1, wherein the sequence of the PTP05polypeptide comprises (A) the full length amino acid sequence of thesequence set forth in (i) SEQ ID NO:5 lacking one or more of thefollowing segments of amino acid residues: 1-187, 188-420, or 421-426;(ii) SEQ ID NO:6 lacking one or more of 44-80, 225-457, or 458-463, or(iii) SEQ ID NO:7 lacking one or more of 1-87, 188-405, or 406-412; (B)(i) residues 1-187, 188-420, or 421-426 of SEQ ID NO:5, (ii) residues44-80, 225-457, 458-463 of SEQ ID NO:6, or (iii) residues 1-87, 188-405,406-412 of SEQ ID NO:7; or (C) the full length amino acid sequence setforth in SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 except that thesequence lacks one or more of the domains selected from the groupconsisting of (i) an N-terminal domain, (ii) a catalytic domain, and(iii) a C-terminal domain.
 9. The antibody or antibody fragment of claim1, wherein the sequence of the PTP10 polypeptide comprises the fulllength amino acid sequence set forth in SEQ ID NO:8 except that thesequence of the PTP10 polypeptide lacks one or more of the domainsselected from the group consisting of (i) an N-terminal domain, (ii) acatalytic domain, and (iii) a C-terminal domain.
 10. The antibody orantibody fragment of claim 1, wherein the antibody is a polyclonalantibody or a monoclonal antibody.
 11. A kit, comprising (i) a firstcontainer containing the antibody of claim 1, and (ii) a secondcontainer containing a conjugate that comprises (a) a binding partner ofthe antibody and (b) a label.
 12. The kit of claim 11, furthercomprising one or more additional containers comprising (i) a washreagent and (ii) one or more reagents for detecting the presence of thebound antibody in a sample.